ES2784271T3 - Specific antibodies for mmp9 - Google Patents

Abstract

An isolated antibody specific for MMP9, or an antigen-binding fragment thereof, wherein said antibody or fragment thereof comprises: (i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, or a variant of SEQ ID NO: 11, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 11: N54Q, N54R, N54K, N54H, D62E , S63T, M116I, D88A and V93L and (ii) further comprising a variable region of the light chain selected from the following group: (1) SEQ ID NO: 24, or a variant of SEQ ID NO: 24, wherein amino acid at position 107 of SEQ ID NO: 24 is substituted with the substitution: V107L; (2) SEQ ID NO: 67, or a variant of SEQ ID NO: 67, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: V45L, V70A and V107L; (3) SEQ ID NO: 29, or a variant of SEQ ID NO: 29, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 29: RsA , V18I, L49M, T62N, L81Q, and A83E; and (4) SEQ ID NO: 69, or a variant of SEQ ID NO: 69, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 69: RsA, V18I, L49M, T62N, L81Q, A83E and FssY.

Description

DESCRIPTION

Specific antibodies for mmp9

Field of the invention

The present invention relates to antibodies and fragments of these that specifically bind and neutralize the activity of said protein, as well as to the uses of these as therapeutic or diagnostic agents.

Background of the invention

The matrix metalloproteinase (MMP) family consists of at least 23 zinc-dependent, structurally related, soluble or membrane-bound endopeptidases, which are widely involved in extracellular matrix remodeling (ECM) and functional regulation of various bioactive molecules.

All MMPs possess a prototype structure that includes a pro-domain that maintains the MMP in an inactive form and a catalytic domain that acts on a wide spectrum of components of the extracellular matrix.

Matrix metallopeptidase 9 (MMP9), also known as 92 kDa type IV collagenase or gelatinase B (GELB), is a member of the MMP family enzymes responsible for the degradation of denatured collagens and basement membrane ( Agrawal et al., 2006 J. Exp. Med. 203, 1007-1019) and to promote inflammation by processing soluble proteins, including protease inhibitors ( Liu et al., 2000, J. Exp. Med. 188, 475- 482), chemokines ( Van den Steen et al., 2000, Lancet Neurol. 2, 747-756), and cytokines ( Nelissen et al, 2003, Brain 126, 1371-1381). MMP9s also control tumor cell migration, invasion, and metastasis through proteolysis of membrane-bound molecules, such as growth factor precursors and receptors, tyrosine kinase receptors (TKR), cell adhesion molecules ( Bauvois, 2012, Biochim Biophys Acta. 1825 ( 1): 29-36). In disease, MMP9 is secreted by many cell types including leukocytes, e.g. g., neutrophils, monocytes / macrophages, and lymphocytes, as well as fibroblasts, myofibroblasts, epithelial cells, smooth muscle cells, endothelial cells, osteoclasts, and tumor cells ( Vandooren et al, 2013, Crit. Rev. Biochem. Mol Biol 48 ( 3 ): 222-72 ).

The general domain structure of MMP9 comprises a secretory leader sequence, an inhibitory pro-domain required for catalytic latency, a 'split' catalytic domain containing three fibronectin type II-like repeat loops that together form a collagen-binding domain (CBD), a hyperglycosylated proline-rich linker (also referred to as the OG domain), and a hemopexin-like C-terminal domain (PEX). Matrix metallopeptidase 2 (MMP2), also known as 72 kDa type IV collagenase or gelatinase A (GELA), is an enzyme that belongs to the same family as MMP9. MMP2 and MMP9 show high amino acid sequence identity (45.9% in the full-length protein and 63.2% in the catalytic domain) and share highly similar 3D structure, especially in their catalytic domain. Therefore, it is very difficult to identify selective anti-human MMP9 inhibitory antibodies against human MMP2 due to its high structural homology and amino acid sequence ( Morgunova et al, 1999, Science, 284: 1667-1670).

Many autoimmune and acute inflammatory disease states, fibrotic states, and invasive cancer are associated with the presence of excess MMP9 ( Hu et al, 2007, Nature Reviews Drug Discovery, 6, 480-498; Ram et al, 2006, J Clin. Immunol., ( 26) 4: 299-307; Ai Zheng, 2003, Chinese journal of cancer, 22 ( 2): 178-84; Baugh et al, 1999, Gastroenterology, 117: 814-822; Santos et al, 2013, Biochem Biophys Res Commun., 438 ( 4): 760-4); Herszenyi et al, 2012, Int. J. Mol. Scl, 13, 13240-13263; Lijnen, 2001, Thromb Haemost, 86: 324-33; Rosell et al., 2005, Stroke 36: 1415-20; Whatling et al., 2004, Arterioscler Thromb Vase Biol., 24: 10-11; Yasmin et al., 2005, Arterioscler Thromb Vase Biol., 25: 372-8; Vassiliadis et al., 2011, BMC Dermatol., 11: 6) , and thus this enzyme has received considerable attention as a prospective target for therapeutic intervention.

Strong clinical and experimental evidence demonstrates the association of elevated MMP9 levels with cancer progression, metastasis, and decreased patient survival, as it plays a key role in tumor cell invasion and metastasis through digestion of the basement membrane and components of the extracellular matrix. Neutrophil gelatinase-associated lipocalin (NGAL), which is covalently bound to MMP9 in human neutrophils ( Triebel et al., 1992, FEBS Lett., 314, 386-388), protects MMP9 against proteolytic degradation, and increases the enzymatic activity of MMP9 and subsequently improves tumor invasiveness and diffusion ( Yan et al., 2001, J. Biol. Chem., 276, 37258-37265). High concentrations of the MMP9 / NGAL complex in serum have been associated with shorter progression-free survival, and overall poor survival in renal cell carcinoma, clear cells ( Perrin et al., 2011, Prog. In Urol J. Assoc. Fr. Urol. Societe Fr. Urol, 21, 851-858).

Specifically, the function of MMP-9 has been associated with colorectal cancer ( Herszenyi et al, 2012, Int J Mol Sci ,. 13 ( 10): 13240-63), pancreatic cancer ( Gao et al, 2015, Med Oncol. 32 ( 1): 418), breast cancer ( Kim et al, 2014, BMC Cancer. 14 ( 1): 959), lung cancer ( Ruiz-Morales et al, Tumor Biol. 36 ( 5): 3601-10) , ovarian cancer ( Naylor et al, 1994, Int J Cancer, 58: 50-6), urinary bladder cancer ( Szarvas et al, 2011, Nat Rev Urol, 8 ( 5): 241-54) and gastric cancer ( Chen et al, 2015, Int J Clin Exp Med. 8 ( 1): 546-57).

The function of MMP9 has been demonstrated in immune pathologies and particularly in inflammatory bowel disease (IBD) where MMP9 has been reported as the most abundantly expressed MMP in actively inflamed intestinal mucosa, and its expression correlates well with disease activity ( Naito and Yoshikawa, 2005, 26: 379-390). Regarding IBD, MMP9 is thought to play a key role in inappropriate tissue remodeling and activation of pro-inflammatory cytokines and chemokines, thereby allowing recruitment of activated leukocytes ( Nuala et al, 2014, Inflamm. Bowel 0: 1 -fifteen). More specifically, the improved expression of MMP9 along the fistula tracts of perianal fistulas and the increased activity of m MP9 in fistula biopsies was reported in patients with Crohn's disease (CD), supporting the hypothesis of that MMP9 may contribute to the formation of fistulas that represent a serious complication of CD ( Efsen, et al, 2011, Basic Clin Pharmacol Toxicol. 109 ( 3): 208-16). Furthermore, decreased serum levels of NGAL / MMP9 also correlate with mucosal healing in ulcerative colitis patients treated with infliximab ( de Bruyn et al, 2014, Inflamm. Bowel Dis., 20, 1198-1207).

The function of MMP9 has been associated with various neurological disorders, for example, Alzheimer's disease ( Mroczko et al, 2013, J. Alzheimers Dis., 37 ( 2): 273-278), multiple sclerosis ( Mirshafiey et al, 2014, Sultan Qaboos Univ Med J., 14 ( 1), 13-25), neuroinflammation or cerebral ischemia ( Candelario-Jalil et al, 2009, Neuroscience 158 ( 3): 983-94). In Alzheimer's disease, the proaggregative influence on the formation of tau oligomers in strategic regions of the brain may be a possible neurotoxic side effect of MMP9 ( Wang et al, 2014 BioMed Res. Int., 2014, ID 908636: 1-8) . It has been suggested that a reduction in mature nerve growth factor (mNGF) as a consequence of high degradation mediated by MMP9, which degrades mNGF in the extracellular space may, in part, justify the pathogenesis of cognitive deficits in mild cognitive impairment and Alzheimer's disease ( Bruno et al, 2009, J Neuropathol. Exp. Neurol., 68 ( 12): 1309-1318).

The function of MMP9 has been associated with fibrotic diseases, for example, systemic sclerosis, multifocal fibrosclerosis, graft-versus-host scleroderma disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, as well as organ-specific disorders, such as pulmonary, liver and kidney fibrosis ( Piera-Velazquez et al, 2011, Am J Pathol, 179 ( 3): 1074-80, Peng et al, 2012, J. Clin. Immunol, 32 ( 6): 1409-14). For example, recent studies have shown that MMPs, in particular MMP9, are involved in the initiation and progression of renal fibrosis through the transition of tubular epithelial cells-mesenchyme and the activation of resident fibroblasts, the transition of epithelial cells. -mesenchyme and pericyte-myofibroblast transdifferentiation ( Zhao et al, 2013, World J Nephrol, 2 ( 3): 84-9).

The pathophysiology of various ocular diseases has been associated with the activity of MMP9. Some examples include: fibrotic pathologies of the lens ( Nathu et al, 2009, Ex. Eye Res., 88 ( 2): 323-330), corneal diseases that are associated with up-regulation of MMP9 ( Sakimoto et al, 2012, Cornea 31, Suppl 1: S50- 6), diabetic retinopathy showing increased levels of MMP9 in patients with disorders of the retina and vitreous humor ( Kowluru et al, 2012, Expert Opin Investig Drugs, 21 ( 6): 797-805 ) and age-related macular degeneration, in which MMP9 was shown to play a role in its pathogenesis ( Nita et al, 2014, Med Sci Monit, 20: 1003-16).

Cardiovascular diseases involve inflammation and altered tissue remodeling associated with the reorganization of the extracellular matrix and the activation of m MP9. Therefore, MMP9 is thought to be associated with the pathophysiology of heart diseases such as hypertension, atherosclerosis, myocardial infarction, heart failure, and coronary artery disease ( Yabluchanskiy et al, 2013, Physiology, 28 ( 6): 391-403 ).

Furthermore, the function of MMP9 has been associated with various groups of disorders such as skin diseases ( Mezentsev et al., 2014, Gene, 540 ( 1): 1-10), sepsis, and acute inflammatory shock syndrome ( Lorento et al, 2014, PLoS One 9 ( 4): e94318; Qui et al. 2012, Comb Chem High Throughput Screen., 15 ( 7): 555-70), osteoarthritis ( Bian et al, 2012, Front Biosci ( Elite Ed ). 4: 74-100), chemotherapy-induced mucositis ( Al-Dasooqi et al, 2009, Cancer Chemother Pharmacol, 64: 1-9), oral diseases ( Al-Azri et al, 2013, Oral Diseases, 19: 347 -359), osteosclerosis ( Teti et al, 1999, J Bone Miner Res. 14 ( 12): 2107-17), endometriosis ( Pitsos et al, 2009, Reprod Scl, 16 ( 8): 717-26) or Chagas ( Geurts et al, 2012, Pharmacol Ther., 133 ( 3): 257-79).

Both monomeric and dimeric forms of MMP9 have been identified in a variety of normal and tumor cells ( Goldberg et al, 1992, J. Biol. Chem., 267, 4583-4591) and in biological fluids and tissues, indicating that both forms are physiologically important. In addition to proteolysis, the dimerization of MMP9 through the hemopexin domain seems to be necessary for better cell migration of MMP9 ( Dufour et al, 2010, J. Biol Chem., 285, 35944-35956), and the study of patterns of MMP9 monomer and dimer secretion in a variety of carcinoma, sarcoma, adenosarcoma and leukemia cell lines revealed that high levels of MMP9, and especially dimers, correlate with the most aggressive cancer cell lines ( Roomi et al, 2014, Int. J. Oncol. 44, 986-992). Taken together, these observations highlight the importance of an effective MMP9 neutralizing agent to efficiently inhibit all natural forms of MMP9, and more specifically, the MMP9 dimer and the NGAL / MMP9 complex to treat highly aggressive metastatic cancers.

Historically, strategies for blocking MMPs have focused on designing small molecule inhibitors that intimately interact with the catalytic site of the activated enzyme. To date, this approach has not been able to translate into the expected clinical benefit, due in part to dose-limiting toxicity and serious side effects, such as musculoskeletal syndrome. Since the MMP9 catalytic site architecture is highly conserved throughout the MMP family, this contra-indication may be attributable to the lack of target selectivity of MMPs at therapeutic doses.

Antibodies or antibody fragments are likely to interact with, and occlude, a much larger portion of the MMP9 structure than active site-directed small molecules, providing greater target inhibitory selectivity.

Some antibodies specific for MMP9 have been described in the prior art, such as mouse AB0041 (WO 2012/027721) and humanized AB0045 (WO 2013/130078), as well as human 539A-M0240-B03 (US 2009/0311245 ; WO 2009/111450; WO 2010/045388), M0166-F10 (US 2009/0311245; US 2011/0135573; WO 2009/111450; WO 2010/045388), 539A-M0237-D02 (US 2009/0297449 and US 2011/0135573), M0279-A03 (WO 2010/045388) mouse REGA-3G12 (WO 02/066057; Martens et al., 2007, Biochim. Biophys. Acta 1770, 178-186). Some of the prior art antibodies have been reported to bind to both MMP9 and MMP2. WO 2011/028883 describes anti-MMP9 antibodies that are specific only for MMP9.

Therefore, there remains a need to develop new therapeutic agents that show high affinity and specificity for MMP9 and have weak or limited affinity and / or specificity to other MMPs such as MMP2, that show improved cross-reactivity to non-human MMP9 orthologs and possess other additional properties, such as reduced immunogenicity in humans, and / or greater stability, which make them particularly suitable for therapeutic applications in humans.

Brief description of the invention

The present invention is directed to proteins that bind to MMP9, in particular human MMP9, and comprise at least one fragment of a variable region of the heavy chain and / or at least one fragment of a variable region of the light chain of a antibody, as described herein.

A first aspect of the invention relates to an isolated antibody specific for MMP9 or antigen-binding fragment thereof, wherein said antibody or fragment binds to ^m MP9 through interaction with an epitope comprising at least one amino acid within a region comprising SEQ ID NO: 41, at least one amino acid within a region comprising DEC ID NO: 42, and at least one amino acid within a region comprising SEQ ID NO: 43, wherein said regions are within the catalytic domain of human MMP9. A second aspect of the invention provides an isolated MMP9-specific antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11 or a variant of SEQ ID NO: 11. NO: 11, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: N53Q, N53R, N53K, N53H, D61E, S62T, M100LI, D84A and V89L. SEQ ID NO: 11 as defined herein above comprises:

(i) a heavy chain CDR1 of SEQ ID NO: 2

(ii) a heavy chain CDR2 of SEQ ID NO: 3

(iii) a heavy chain CDR3 of SEQ ID NO: 4

A more specific aspect of the invention provides an isolated antibody, as already described, which further comprises a variable region of the light chain selected from the following group:

(1) SEQ ID NO: 24, or a variant of SEQ ID NO: 24 with the V104L substitution;

(2) SEQ ID NO: 67, or a variant of SEQ ID NO: 67, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: V46L, V71A and V104L; (3) SEQ ID NO: 29, or a variant of SEQ ID NO: 29, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: R8A, V19I, L47M, T60N, L79Q, and A81E; Y

(4) SEQ ID NO: 69, or a variant of SEQ ID NO: 69, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: R8A, V19I, L47M, T60N, L79Q, A81E and F86Y.

SEQ ID NO: 24 and SEQ ID NO: 67 as defined above comprise:

(i) a light chain CDR1 of SEQ ID NO: 21

(ii) a light chain CDR2 of SEQ ID NO: 22

(iii) an acidic light chain CDR3 of SEQ ID NO: 23

SEQ ID NO: 29 and SEQ ID NO: 69 as defined above comprise:

(i) a CDR1 of the light chain of SEQ ID NO: 26

(ii) a light chain CDR2 of SEQ ID NO: 27

(iii) a CDR3 of the light chain of SEQ ID NO: 28

A third aspect of the invention relates to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof as described herein.

A fourth and fifth aspects of the invention relate to a recombinant expression vector comprising said nucleic acid molecule, and a host cell comprising said recombinant vector, respectively. A sixth aspect of the invention provides a pharmaceutical composition comprising one or more of the following: (i) an MMP9-specific antibody or antigen-binding fragment thereof, (ii) a nucleic acid sequence, (iii) a vector and / or (iv) a host cell, as described herein, and at least one pharmaceutical acceptable carrier.

A seventh aspect of the invention refers to an antibody or formulation thereof, according to the invention, for use in the prevention and / or treatment of a disorder related to MMP9, such as an inflammatory and / or autoimmune disease, or a cancer or tumor, or a fibrotic disease.

Another aspect of the invention relates to an anti-MMP-9 antibody of the invention, or a formulation thereof, for use as a medicine.

Other features and advantages of the invention will be apparent from the following detailed description.

Description of the figures

Figure 1 shows a schematic diagram of the structure of the molecular domain of human MMP9 protein. The numbers indicate the amino acid positions in the amino acid sequence of the immature MMP9 protein.

Figure 2 shows the alignment of the amino acid sequences of human, Cynomologus monkey (cyno), rat and mouse MMP9. The (*) indicates the positions that have a single fully conserved residue, (:) indicates the conservation between groups of strongly similar properties - with a score> 0.5 in the Gonnet PAM 250 matrix, (.) Indicates the conservation between groups of weakly similar properties similar - with a score <0.5 in the Gonnet PAM 250 matrix.

Figure 3 shows the alignment of the sequences of exemplary human heavy chain antibody fragments comprising the human heavy chain constant region and the human heavy chain variable region of anti-MMP9 antibodies, according to the invention. The sequences of the heavy chain antibody fragments are tagged with the variable heavy chain region sequence name with * before the name: * F20-VH, * F20-VH-GL1, * F20-VH- GL1-V1-V9, * F20-VH-GL1-V1-V9-V14, * F20-VH-GL1-V4-V9, * F20-VH-GL1-V4-V9-V14. CDRs are underlined. The annotations are identical to those described in Figure 2.

Figure 4 shows the sequence alignment of the exemplary human light chain antibody fragments comprising the human light chain constant region and the human light chain variable region of anti-MMP9 antibodies, according to with the invention. The sequences of the light chain antibody fragment are tagged with the variable light chain region sequence name with * before the name: * B03-VL, * B03-VL-GL1. CDRs are underlined. The annotations are identical to those described in Figure 2.

Figure 5 shows the sequence alignment of exemplary human light chain antibody fragments comprising the human light chain constant region and the human light chain variable region of anti-MMP9 antibodies according to with the invention. The sequences of the light chain antibody fragment are labeled with the variable light chain region sequence name included with * before the name: * B08-VL, * B08-VL-GL6. CDRs are underlined. The annotations are identical to those described in Figure 2.

Figure 6 shows the titration of the neutralizing activity of the exemplary anti-MMP9 antibodies, according to the invention (the variants F20-VH / B03-VLc and F20-VH / B08-VLc) towards human proMMP9 (A, B) and mature human MMP9 (C, D). Variants F20-VH / B03-VLc (A, C), Variants F20-VH / B08-VLc (B, D).

Figure 7 shows the neutralizing activity of exemplary anti-MMP9 antibodies according to the invention towards the catalytic domains of various recombinant human matrix metalloproteinases.

Figure 8 shows the titration of the neutralizing activity of the exemplary anti-MMP9 antibody according to the invention (the F20-VH / B08-VLc variant; solid lines and dark symbols) or isotype control (broken lines and light symbols) towards MMP9 Neutrophil-derived human dimeric (circles), human monomeric MMP9 (triangles), and the human NGAL-MMP9 complex (squares).

Figure 9 shows the titration of the neutralizing activity of the exemplary anti-MMP9 antibody according to the invention (variant F20-VH / B08-VLc; solid lines and dark symbols) or the comparative anti-MMP9 antibody 1 (broken lines and symbols light) towards human monomeric MMP9 activated with MMP3 (triangles), dimeric MMP9 (circles), and NGAL-MMP9 complex (squares) derived from human neutrophils.

Figure 10 shows the kinetics of binding of anti-MMP9 antibodies to recombinant human MMP9 antigen. MMP3-activated MMP9 (panels A and B) and pro-MMP9 (panels C and D) were titrated on a BIAcore sensor chip in which anti-MMP9 antibodies, the F20-VH / B08-VLc variant (panels A and C) and comparative antibody 1 (panels B and D), have been immobilized first. The sensorgrams of the binding kinetics are reported. Resonance unit (RU). All y axes are Response (RU); all x axes are Time.

Figure 11 shows the direct and competitive binding of anti-MMP9 antibodies to human MMP9. The direct binding capacity of various concentrations of the F20-VH / B08-VLc variant of biotinylated anti-MMP9 antibodies (dark circles) and comparative antibody 1 (light circles) towards pro-MMP9 (panel A) or activated MMP9 with MMP3 (panel B) were evaluated using a normal ELISA protocol. The MMP9 antigens were coated at a concentration of 2.5 pg / mL. Controls were streptavidin-HRP alone (dark triangles), or human IgG4 isotype control plus biotinylated anti-human IgG second pass Fab antibody (dark squares).

For competitive binding experiments (panel C), after pre-incubation with comparative antibody 1, the purified F20-VH / B08-VLc variant (both used at 50 pg / mL), or buffer (no antibody) was revealed coated MMP3-activated MMP9 using an optimal dose of biotinylated comparative antibody 1 (2.5 pg / mL) or the F20-VH / B08-VLc variant (50 pg / mL). For panels A, B, C, the results are expressed as the mean ± SD (standard deviation) of the corrected absorbance (A450-A620) for each condition performed in duplicate.

Figure 12 shows the effect of the anti-MMP9 antibody (F20-VH / B08-VLc variant) on cancer cell line invasion across a Matrigel-coated Transwell® culture plate. MGC803 human gastric cancer cells were incubated with phorbol 12-myristate 13-acetate (PMA) and with a general chemical inhibitor of MMPs (GM-6001), with the anti-MMP9 antibody (F20-VH / B08-VLc) , the isotype control or the medium only. Invasive cells were quantified after 16 hours using Calcein-AM. Each condition was performed in duplicate and the means and SD of the fluorescence units were reported.

Figure 13 shows endoscopic scores in a DSS-induced mouse colitis model. On day 6 after induction with DSS (sodium dextran sulfate), the mice were treated with the anti-MMP9 antibody (F20-VH / B08-VLc variant) or with the isotype control antibody. The effects of the F20-VH / B08-VLc variant are shown as black bars, the isotype control is shown as shaded bars. The means and SD of the endoscopic scores for both groups of treated mice were reported (n = 5 for F20-VH / B08-VLc; n = 6 for the isotype control); Statistical comparisons of group data (F20-VH / B08-VLc versus isotype control on day 14 after DSS induction) were performed using the bidirectional, unpaired T-test using Graph Pad Prism. * p = 0.005.

Figure 14 shows the total histological scores, infiltration scores and epithelial damage of colon sections of mice with DSS-induced colitis, treated with the anti-MMP9 antibody (F20-VH / B08-VLc variant) or the control antibody. isotype. (A) Total histological score, (B) infiltration (C) epithelial damage. Individual values, means and SDs for each criterion are reported for both groups of treated mice (n = 5 for F20-VH / B08-VLc; n = 5 for the isotype control).

Figure 15 shows representative cross-sections of explanted mouse intestinal grafts stained with hematoxylin-eosin. (A) Day 0 - Resection of freshly isolated small intestine with open lumen and typical crypt structures. (B) Isotype control day 14 - complete occlusion of the intestinal lumen 14 days after transplantation in the mouse treated with isotype control (n = 5). (C) Variant F20-VH / B08-VLc day 14 - partial occlusion of the intestinal lumen 14 days after transplantation in mice treated with anti-MMP9 (n = 5).

Figure 16 shows representative cross sections of explanted mouse intestinal grafts stained with Sirius red and quantification of collagen layer thickness. Freshly isolated small intestine resection (A and B). Day 14 after transplantation in mice treated with the isotype control (C and D). Day 14 after transplantation in mice treated with anti-MMP9 antibody (F20-VH / B08-VLc variant) (E and F). Transmission light (A, C and E), polarized light (B, D and F). (G) Quantification of collagen layer thickness in heterotopic intestinal grafts from mice treated with anti-MMP9 or isotype control. The means and SD of the thickness of the collagen layer of the transplant sections are reported for both groups of treated mice (72 sections for F20-VH / B08-VLc; 64 sections for the isotype control). Statistical comparisons of the group data (F20-VH / B08-VLc versus the isotype control) were made using the unpaired, bidirectional t-test, using Graph Pad Prism. *** p <0.0001.

Detailed description of the invention

Definitions

The term "Matrix metalloproteinase 9", abbreviated as "MMP9", also known as 92 kDa type IV collagenase, gelatinase or 92 kDa gelatinase B (GELB), is an enzyme that, in humans, is encoded by the MMP9 gene the sequence of which is described in NCBI accession number ENSG00000100985. The human MMP9 form comprises a sequence of 707 amino acids in total, available under the accession number NCBI NP 004985.2 (SEQ ID NO: 1). The general domain structure of MMP9 comprises a secretory leader sequence (residues 1-19 in SEQ ID NO: 1), an inhibitory pro-domain required for catalytic latency (residues 20-106 in SEQ ID NO: 1), a 'split' catalytic domain (residues 107-441 in SEQ ID NO: 1) containing three fibronectin type II-like repeat loops that together form a collagen-binding domain (CBD), a linker rich in hyperglycosylated proline (also referred to as the OG domain) (residues 442-520 in SEQ ID NO: 1), and a C-terminal hemopexin-like repeat domain (PEX) (residues 521-707 in SEQ ID NO: 1) ( Rowsell et al, 2002, J Mol Biol 319: 173-81) (Figure 1). MMP9 is secreted and maintained as a latent, inactive form by a pro-domain. Proteolytic removal of the pro-domain triggers the enzymatic activity of MMP9, and then MMP9 can be referred to as "active MMP9". Its catalytic domain contains a gelatin-binding region, which provides specific affinity for gelatin. In addition to gelatin, MMP9 has several substrates such as collagens (eg, collagen IV and collagen V), elastin, galectin3, entactin, and ICAM-1 ( Ram et al, 2006, J. Clin. Immunol. 26 , 299-307), and cytokines and chemokines ( Opdenakker et al, Trends Immunol. 2001: 22: 527-81). The term "antibody" as referred to herein, designates a polypeptide that binds to an antigen. This includes whole antibodies and any antigen-binding fragments.

The term "antibody" is used in its broadest sense and includes monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and other engineered antibodies, provided that the characteristic properties of the invention are preserved, in particular the ability to binding to the target antigen, more specifically to the same epitope of MMP9, as recognized by the antibodies of the invention. Examples of antibodies and fragments thereof include a variable domain fragment ("Fv", comprising the VH and VL domains of a single arm of an antibody), the Fab fragment (monovalent fragment comprising the VH, VL, CH1 domains and CL), Fab ² fragment (bivalent), Fab3 fragment (trivalent), Fab 'fragment (Fab with hinge region), F (ab') ² fragment (bivalent fragment that includes two Fab fragments linked to a disulfide bridge in the region hinge), Fd fragment (comprising the VH and CH1 domain), rIgG (reduced IgG or semi-IgG), diabodies, tribodies, tetrabodies, minibodies, monovalent antibodies, divalent or multivalent antibodies that comprise a fragment of more than one antibody, Single chain variable fragment (ScFv), bis-scFv (bispecific), and antibody derivatives such as disulfide stabilized Fv fragments, CDR-comprising peptides, as well as epitope-binding fragments of any of the above ( Holliger and H Udson, 2005, Nature Biotechnology, 23 ( 9): 1126-1136). An antibody refers to a glycoprotein comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain comprises a variable region of the light chain (VL) and a constant region of the light chain (CL). In mammals, the heavy chain can be alpha (a), delta (6), epsilon (£), gamma (y) or mu (p), which defines the class of antibody IgA, IgD, IgE, IgG and IgM, respectively. . In mammals, the light chain can be lambda (A) or kappa (k). In mammals, depending on the class of antibody, the heavy chain constant region comprises three immunoglobulin domains, CH1, CH2 and CH3 (for IgA, IgD, IgG) or four immunoglobulin domains, CH1, CH2, CH3 and CH4 ( for IgE and IgM). The constant region of the light chain comprises an immunoglobulin domain, CL. An antibody can have the structure of an IgA, IgG, IgE, IgD and IgM as well as any subtype of these. The antibodies can be of any origin including, in particular, primate (human and non-human primate) and primatized origin.

The term "variable domain" or "variable region" (variable domain of a light chain (VL), variable domain of a heavy chain (VH) as used herein refers to each of the domain pairs of each light and heavy chain that are directly involved in the binding of the antibody to the antigen The variable domains of the light and heavy chain have the same general structure, and each domain comprises four framework regions ("FR") whose sequences are widely conserved, connected by three "hypervariable regions" called "complementarity determining regions" or "the CDRs." The framework regions adopt a p-sheet conformation and the CDRs can form loops that connect the p-sheet structure. The CDRs in each chain are held in place. three-dimensional structure by the framework regions and form, together with the CDRs of the other chain, the antigen-binding site. The term "antigen-binding portion of an antibody" when used herein refers to the amino acid residues of an antibody that are responsible for antigen-binding. The antigen-binding portion of an antibody comprises amino acid residues from the "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those regions of the variable domain other than the hypervariable region residues as defined herein. Therefore, the variable domains of the light and heavy chain of an antibody comprise from N- to C-terminus: the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The residues in the CDR and FR regions are numbered in a conventional manner according to the standard definition of Kabat et al. ( Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ( 1991), Publication No. 91-3242). This numbering system is used in this specification except where otherwise indicated. The Kabat residue designations do not always correspond directly to the linear numbering of amino acid residues of. The actual, linear amino acid sequence may contain fewer or more amino acids than the strict Kabat numbering corresponding to a shortening, or insertion into, a structural component, either of the framework or complementarity determining region (CDR), of the structure. of the basic variable domain. Correct Kabat residue numbering can be determined for a given antibody by aligning residues of homology in the antibody sequence with a "normal" Kata numbering sequence. The heavy chain variable domain CDRs are located at residues 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. The light chain variable domain CDRs are located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2) and residues 89-97 (CDR-L3) according to the Kabat numbering system.

In the present application, unless otherwise specified, for all human immunoglobulin heavy and light chain variable domains, the numbering is in accordance with the "Kabat numbering system" ( Sequences of Proteins of Immunological Interest, 5a ed., Public Health Service, National Institutes of Health, Bethesda, MD ( 1991), Publication No. 91-3242 ).

In the present application, unless otherwise specified, for all human immunoglobulin heavy chain constant domains, numbering is according to the "EU numbering system" ( Edelman et al, 1969, Proc Natl Acad Sci, 63 ( 1): 78-85).

The term "monoclonal antibody" when used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, each of the antibodies that comprise the population are identical, except for possible naturally occurring mutations that they may be present in smaller quantities. Monoclonal antibodies are highly specific, being directed against a single antigenic site. The modifier "monoclonal" indicates the character of the antibody that was obtained from a fairly homogeneous population of antibodies, and should not be construed as requiring the production of the antibody by any particular method.

The term "chimeric antibody" generally refers to an antibody that comprises a variable region from one source or species and at least a part of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. A typical example of chimeric antibodies includes those that comprise a murine variable region and a human constant region. As defined herein, this term also includes an antibody that comprises at least one of the CDRs of a first human antibody and at least a portion of a constant region of a second human antibody. It should include an antibody that comprises CDR1, CDR2 and CDR3 of the heavy chain of a first human antibody and CDR1, CDR2 and CDR3 of the light chain of a second human antibody.

The term "humanized antibody" designates antibodies from a non-human species that have one or more complementarity determining regions (CDRs) from said non-human species and a framework region from a human immunoglobulin molecule. Humanized antibodies may, as an option, further comprise one or more framework residues derived from the non-human species from which the CDRs were derived.

The term "human antibody" or "fully human antibody" refers to antibodies in which the variable regions and constant regions of both heavy and light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily of the same antibody.

The term "isolated antibody" refers to an antibody that has been separated from a component of its natural environment. For example, an isolated antibody has been purified to greater than 95% or 99% purity, as determined by methods known in the art (see, eg, Flatman et al, 2007, J Chromatogr B Analyt Technol Biomed Life Sci, 848: 79-87) including electrophoretic (eg, SDS-PAGE, isoelectric focusing, capillary electrophoresis) or chromatographic (eg, ion exchange or HPLC (high performance liquid chromatography) phase reverse

The terms "polynucleotide" or "nucleic acid molecule" refer to a polymer that comprises nucleotides. Examples of nucleic acid molecules include DNA, RNA, blocked nucleic acid (LNA), complementary DNA (cDNA).

"Polypeptide" is understood as a peptide, an oligopeptide, an oligomer or a protein comprising at least two amino acids linked together by a normal or modified peptide bond, as may be in the case of isosteric peptides, for example. A polypeptide can be composed of amino acids other than the 20 amino acids defined by the genetic code. A polypeptide can also be composed of amino acids modified by natural processes, such as post-translational maturation processes or by chemical processes, which are well known to a person skilled in the art. Such modifications are fully detailed in the literature. These modifications can appear in any part of the polypeptide: in the peptide backbone, in the side chain or even in the carboxy- or amino-terminal ends. For example, modifications of the polypeptide are understood to include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of heme, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or of a lipid derivative, covalent fixation of a phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, cysteine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation including pegylation, GPI anchor formation, hydroxylation , iodination, methylation, myristoylation, oxidation, proteolytic processes, phosphorylation, prenylation, racemization, seneloylation, sulfation, addition of amino acids such as arginylation or ubiquitination. Such modifications are fully detailed in the literature ( Proteins Structure and Molecular Properties ( 1993) 2nd Ed., TE Creighton, New York; Post-translational Covalent Modifications of Proteins ( 1983) BC Johnson, Ed., Academic Press, New York; Seifter et al. ( 1990) Analysis for protein modifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 and Rattan et al., ( 1992) Protein Synthesis: Post-translational Modifications and Aging, Ann NY Acad Sci, 663: 48 -62).

"Isolated polynucleotide" or "isolated polypeptide" is understood as a polynucleotide or a polypeptide as defined above, which is isolated from a human body or is otherwise produced by a technical process.

The term "variant" can apply to a polynucleotide and / or a polypeptide. For example, a variant of a peptide or polypeptide, as mentioned herein, means a peptide or polypeptide that is considerably homogeneous to the referenced peptide sequence, but which has a different amino acid sequence from the referenced sequence. refers to due to one or more amino acid deletions, insertions and / or substitutions. "Considerably homologous" means a variant amino acid sequence that is identical to the referenced peptide sequence except for deletion, insertion and / or substitution of a few amino acids, e.g. eg, 1, 2, 3, 4, 5, or 6 amino acids. Considerably homologous means a variant amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence referred to. A variant nucleic acid sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, or at least 97%, at least 98%, or at least 99% identical to the nucleic acid sequence referred to. The identity of two amino acid sequences or of two nucleic acid sequences can be determined by visual inspection and / or mathematical calculations, or more easily by comparing the sequence information using the known computer program used for sequence comparison such as the Clustal package version 1.83. A variant may comprise a sequence that has at least one conservatively substituted amino acid, meaning that a particular amino acid residue is replaced by a residue that has similar physicochemical characteristics. In general, substitutions for one or more amino acids present in the parent polypeptide must be made conservatively. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for each other, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other such conservative substitutions, for example whole region substitutions having similar hydrophobicity characteristics, are well known ( Kyte, et al, 1982, J. Mol. Biol, 157: 105-131). For example, a "conservative amino acid substitution" may involve a substitution of a native amino acid residue with a non-native residue in such a way that there is little or no effect on the polarity or charge of the amino acid residue at that position. Desired amino acid substitutions (conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. Exemplary amino acid substitutions are presented in Table 1 below. The term "variant" also includes a peptide or polypeptide considerably homologous to the referenced peptide sequence, but having a different amino acid sequence from the referenced sequence because one or more amino acids have been chemically modified or substituted by amino acid analogs. This term also includes glycosylated polypeptides.

Table 1

The term "epitope" includes any antigenic determinant capable of specifically binding an antibody or antigen-binding fragment thereof. In certain embodiments, the epitope determinant includes chemically active surface groupings of molecules such as amino acids, sugar, phosphoryl, or sulfonyl side chains, and in certain embodiments, they may have specific three-dimensional structural characteristics and / or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. Some epitopes comprise discontinuous sections of the antigen's amino acid sequence, where non-contiguous amino acids are positioned close to each other by the antigen's spatial configuration ("conformational epitopes") or comprise a contiguous amino acid section in the antigen's amino acid sequence. ("linear epitopes").

When used herein the term "binding" or "binding" of an antibody to a target antigen means at least temporary interaction or association of said antibody with, or, said target antigen (such as MMP9) or with, or , fragments of said target antigen that comprise an epitope recognized by said antibody.

The terms "selectively binds", "specifically binds", "specific for", when applied to an antibody, indicate that the antibody preferentially recognizes and / or binds to the target polypeptide or epitope, ie, with higher affinity than to any other antigen or epitope, that is, binding to the target polypeptide can be discriminated from non-specific binding to other antigens. The binding affinity of an antibody can be readily determined by one skilled in the art, for example, by Scatchard analysis ( Scatchard et al., 1949, Ann. NY Acad. 1949. 51, 660-672).

When used herein, "binding affinity" generally refers to the apparent association constant or "Ka". Ka is the reciprocal of the dissociation constant "Kd". Binding affinity can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, resonance, or surface plasmon spectroscopy (eg, using a fluorescence assay). Exemplary conditions for the evaluation of binding affinity are in the TRIS buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCb at pH 7.5). These techniques can be used to measure the concentrations of the bound and free binding protein as a function of the concentration of the binding (or target) protein. The concentration of bound binding protein ([Bound]) is related to the concentration of free binding protein ([Free]) and the concentration of binding sites for the binding protein on the target, where (N) is the number of binding sites per target molecule by the following equation: [Bound] = N x ([Free]) / ((1 / Ka) [Free]). The affinity comparison between two antibodies can be established without actually determining the Ka value for each antibody, but based on a quantitative affinity measurement (eg, by ELISA or FACS analysis) that is proportional to Ka or a measurement qualitative affinity or an inference of affinity (eg, in functional assays or in vitro or in vivo assay ).

The term "blocking" or "neutralizing" activity of an antibody refers to its ability to inhibit the activity of its target. Applied to an antibody that binds to MMP9, this term refers to the ability of the antibody to neutralize, in general, the activity of MMP9, but inhibiting the activation of proMMP9 and / or inhibiting the catalytic activity of activated MMP9 on one of its substrates such as gelatin, for example, as described in the Examples section . The neutralizing activity of an anti-MMP9 antibody can be determined by cell-free in vitro assays, or in in vivo assays or in vitro functional assays such as an invasion assay of human cancer cell lines. In a transwell invasion assay of human cancer cell lines, cancer cells degrade and migrate through a basement membrane matrix (Matrigel®), thus limiting the in vivo process of intravasation of tumor cells into vessels. nearby blood vessels and extravasation and invasion into distant tissue.

The "potency" of an antibody can be expressed as the concentration of antibody / antigen-binding fragment that produces the maximum mean effect at a given antigen concentration. For example, the "effect" of an antibody may be the inhibition or neutralization of the activity of its target. In this case, the antibody concentration that produces the maximum mean inhibition is called IC ⁵⁰ , which is given in mol / L or M. Potency is normally influenced by affinity until, at a given antigen concentration, an affinity is it reaches beyond which other improvements in affinity will no further improve antigen binding (called potency cap or maximum potency). Applied to an antibody against MMP9, the potency can, for example, be determined by measuring the IC ⁵⁰ value of the MMP9-dependent digestion of the gelatin substrate in the presence of the antibody.

The term "inhibition efficacy" or "neutralization efficacy", applied to a neutralizing antibody, is a measure of the efficacy of said antibody in inhibiting a specific biological or biochemical function expressed as a percentage of the total potential inhibition of the biological or biochemical activity that is normalized to 100%. Applied to an antibody that binds to MMP9, 100% efficiency may, for example, be total antibody-mediated inhibition of MMP9-dependent digestion of a gelatin substrate. The term "effector function" of the antibody, when used herein, includes a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include FcyR-mediated effector functions such as ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell-mediated phagocytosis), and complement-mediated effector functions such as CDC (complement-dependent cytotoxicity). ). An effector function of an antibody can be altered by changing, ie improving or reducing, preferably enhancing, the affinity of the antibody for an effector molecule such as an Fc receptor or complement component. The binding affinity of an Fc region of the antibody to an Fc receptor or ligand can be altered by modifying the binding site of the effector molecule. It is also possible that an alteration in the binding site on the antibody for the effector molecule alters the geometry of the interaction without significantly altering the overall binding affinity, rendering the effector mechanism ineffective as in non-productive binding. It is also possible to alter an effector function by modifying a site not directly involved in the binding of the effector molecule, but otherwise implicating the performance of the effector function. By altering the effector function of an antibody it may be possible to control various aspects of the immune response, e.g. eg, enhancing or suppressing various reactions of the immune system, with possible beneficial effects in diagnosis and therapy.

The term "pharmaceutically acceptable" refers to a carrier comprising a material that is not biologically or otherwise undesirable.

The term "carrier" refers to any component present in a pharmaceutical formulation other than the active agent and thus includes diluents, binders, lubricants, disintegrators, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives. and the like.

When used herein, "treatment" and "treating" and the like, in general, means obtaining a desired pharmacological or physiological effect. The effect may be prophylactic in terms of the prevention or partial prevention of a disease, symptom or condition thereof and / or it may be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease. The term "treatment" when used herein covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in an individual who may be predisposed to the disease but still it has not been diagnosed as having it, for example, based on family history; (b) inhibiting the disease, that is, arresting its development; or (c) alleviating the disease, that is, causing regression of the disease and / or its symptoms or conditions such as improvement or repair of the damage. For example, the treatment of inflammatory bowel disease comprises the prevention, reduction or even eradication of the symptoms of diseases or disorders, for example, the partial or total relief of diarrhea, abdominal pain and cramps, blood in the stool, abscesses , ulcers and fistulas.

The term "MMP9-related disorders", as defined herein, designates diseases mediated or influenced, at least in part, by MMP9 expression and / or activity. Examples of MMP9-related diseases include inflammatory and autoimmune diseases, cancers or tumors, lung diseases, fibrotic diseases such as fibrotic lung diseases, septicemia, dystrophy. muscle, allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and eye diseases.

The terms "inflammatory and autoimmune diseases" are defined herein, generally, as inflammatory abnormalities that may or may not involve the immune system and diseases or disorders that arise from an abnormal immune response of the individual's body against substances and tissues normally present in the body, respectively. Non-limiting examples of inflammatory and autoimmune diseases mainly include inflammatory bowel diseases (IBD) including Crohn's disease (CD) (in particular Crohn's disease with penetrating (fistulizing) and constrictive (stenosing) consequences, ulcerative colitis (UC), colitis indeterminate, collagenous colitis, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, polymyositis, atherosclerosis.

The term "inflammatory bowel disease" (IBD) is defined herein as a disease involving chronic inflammation of all or part of the digestive tract. Non-limiting examples of IBD include Crohn's disease, in particular non-penetrating (non-fistulizing) and non-constrictive (non-stenosing) Crohn's disease, penetrating (fistulizing) and constrictive (stenosing) Crohn's disease and fistulizing Crohn's disease, ulcerative colitis (UC), indeterminate colitis, collagenous colitis, lymphocytic colitis and intestinal fibrosis. Crohn's disease (CD) is defined as a disease of transmural inflammation with irregular (patchy) lesions that can involve any part of the GI tract from the mouth to the anus. Ulcerative colitis is a disease of mucosal inflammation limited to the colon.

The terms "cancers" or "tumors" as defined herein, are diseases that involve abnormal cell growth with the potential to invade or spread to other parts of the body. The term "cancers" designates diseases exemplified by, but not limited to, hematopoietic cancer, brain cancer, breast cancer, colorectal cancer, head and neck cancer, pancreatic cancer, ovarian cancer, urinary bladder cancer, lung cancer. , liver cancer, melanoma, prostate cancer, muscle cancer, mesenchymal cancer, esophagogastric adenocarcinoma, non-small cell lung cancer, squamous cell lung carcinoma, lung adenocarcinoma, gastric adenocarcinoma, pancreatic adenocarcinoma, hepatocellular carcinoma, and colorectal cancer.

The term "lung disease" designates diseases exemplified by, but not limited to, asthma, fibrotic lung diseases such as idiopathic pulmonary disease, chronic obstructive pulmonary disease (COPD), and rhinitis.

The term "fibrotic diseases" is defined herein as a disease in which the affected tissues present an excessive accumulation of fibrous connective tissues (components of the extracellular matrix such as collagen and fibronectin) in and around the inflamed or damaged tissue, which It can lead to permanent scarring, organ malfunction, and ultimately death, as seen in end-stage liver disease, kidney disease, idiopathic pulmonary fibrosis (IPF), and heart failure. Non-limiting examples of fibrotic diseases include systemic sclerosis, multifocal fibrosclerosis, graft versus host disease with sclerodermal complication in bone marrow transplant recipients, nephrogenic systemic fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus.

The terms "ocular conditions" or "ocular diseases" are defined herein as eye diseases associated with progressive degeneration of the retinal pigment epithelium and photoreceptors that give rise to visual loss and / or eye diseases associated with damage to the eye. blood vessels of the retina. Non-limiting examples of ocular conditions include fibrotic pathologies of the lens, corneal diseases, diabetic retinopathy, "dry" or "wet" age-related macular degeneration, proliferative vitreoretinopathy, cataract formation, pterygium, keratoconus, macular degeneration related to age and diabetic retinopathy.

The term "cardiovascular diseases" is defined herein as diseases of the cardiovascular system involving inflammation, impaired remodeling of tissue with increased collagen and accumulation of fibrotic scar in myocardial infarction. Non-limiting examples of cardiovascular diseases include hypertension, pulmonary hypertension, pulmonary or tricuspid valve disease, aortic and mitral valve disease, aortic coarctation, atherosclerosis, myocardial infarction, heart failure, diabetic cardiomyopathy, dilated cardiomyopathy, chronic arrhythmia, cardiac fibrosis and coronary artery disease.

The terms "neurological disorders" or "neuropsychiatric disorders" are defined herein as diseases characterized by neuronal dysfunction and neuronal cell death, resulting in incurable and often fatal functional deficits. Non-limiting examples of neurological disorders include amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, neuronal inflammation, cerebral ischemia, and neuropathic pain.

The term "individual" when used herein refers to mammals. For example, mammals contemplated by the present invention include humans, primates, domesticated animals such as cattle, sheep, pigs, horses, laboratory rodents, and the like.

The term "efficacy" of a treatment or method according to the invention can be measured based on changes in the course of the disease or condition in response to a use or a method according to the invention. For example, the efficacy of a treatment or method according to the invention can be measured by its impact on the signs or symptoms of the disease. A response is achieved when the patient experiences partial or complete relief, or reduction of the unwanted symptoms of the disease.

The term "effective amount" when used herein refers to an amount of at least one antibody according to the invention, or a pharmaceutical formulation thereof, that causes a detectable reduction in symptoms of the disease in an individual upon given such an antibody, these symptoms may include, for example: a) diarrhea, abdominal pain and cramps, blood in the stool, abscesses, ulcers and fistulas, in the case of inflammatory bowel disease or b) constipation or diarrhea, blood bright red or dark red stools, weight loss, fatigue, nausea and anemia, in the case of colorectal cancer.

MMP9 binding proteins

General characteristics of MMP9-binding proteins

In a first aspect, the present invention provides proteins that bind to MMP9, in particular human MMP9, or a fragment thereof, and comprise at least one fragment of a variable region of the heavy chain and / or at least one fragment of a variable region of the light chain of an antibody as described herein.

In one embodiment of the invention isolated antibodies specific for MMP9, in particular human MMP9, or antigen-binding fragments thereof are provided, which comprise at least one fragment of a variable region of the heavy chain and at least one fragment of a region light chain variable, and as an option, at least a fragment of a constant region, as described herein.

In an alternative embodiment of the invention isolated antibodies specific to MMP9, in particular human MMP9, antigen-binding fragments thereof, characterized by binding to an epitope on MMP9, are provided, as described herein.

The protein to which the antibodies according to the invention bind, or fragments thereof, can be the MMP9 protein of any species.

Antibodies according to the present invention generally have high specificity for human MMP9. However, depending on the degree of sequence identity between MMP9 homologs from different species (see Figure 2), a given antibody or antigen-binding fragment may show cross-reactivity with MMP9 from at least one other species, e.g. eg, mouse, rat, marmoset, monkey (eg, Cynomologus monkey), dog, and / or rabbit. For antibodies directed toward human MMP9, some level of cross-reactivity with other mammals, some level of cross-reactivity with other forms of mammalian MMP9 may be desirable in certain circumstances, for example, when testing antibodies in animal models of a particular disease or for conduct toxicology, safety, and dosage studies.

In a specific embodiment, antibodies according to the invention or fragments thereof preferentially bind to a human MMP9.

In another embodiment, the antibodies according to the invention, or fragments thereof, show cross-reactivity with human MMP9, Cynomologus monkey MMP9, rat MMP9, and as an option, mouse MMP9.

In some embodiments, the binding affinity (eg, inversely correlated with the Kd value) of antibodies and fragments thereof, according to the invention for human MMP9 is at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than its binding affinity for a non-human MMP9.

In one embodiment, the antibodies according to the invention or fragments thereof bind preferentially to MMP9 and, as an option, further exhibit weak, or virtually non-binding (i.e. negligible or undetectable binding) to other metalloproteinases of matrix (MMP) such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, MMP13, MMP14, MMP16, MMP17, MMP19.

In a particular embodiment, the antibodies according to the invention, or fragments thereof, preferentially bind to MMP9 and exhibit weak, or virtually non-binding (ie negligible or undetectable) binding to MMP2.

For therapeutic uses, it may be advantageous that the antibodies according to the invention, or fragments thereof, do not bind, and thus do not neutralize, MMP2 so as not to significantly affect the activity of MMP2. In fact, MMP2 is required for normal tissue homeostasis and may also have a protective function against disease, as suggested by observations that mice with the silenced MMP2 gene show a worse phenotype than native-type mice, in various disease models ( Grag et al., 2006, J. Immunol, 177 ( 6): 4103-12).

In some embodiments, the binding affinity of antibodies (eg, inversely correlated with the Kd value of the equilibrium dissociation constant), and fragments of these, according to the invention for MMP9 is at least 2 times, at at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than your binding affinity for MMP2.

Binding affinity can be measured by any method known in the art including dialysis including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, resonance, or surface plasmon spectroscopy (eg, using a fluorescence assay) ( Jiang et al. BMC Pharmacology 2010, 10:10) and can be expressed as, for example, association constant (on-rate), dissociation constant (off-rate), dissociation constant (Kd), equilibrium constant (Keq ) or any other term used in the art.

In some embodiments, antibodies, and fragments thereof, according to the invention specifically bind to human MMP9 with a dissociation constant (Kd) equal to or less than 100 nM, in particular less than 10 nM, more particularly less than 1 nM, or less than 0.5 nM, or less than 0.1 nM, or less than 0.01 nM, or less than 0.005 nM. The protein to which the antibodies according to the invention, or fragments thereof, bind is any form of MMP9: the immature protein comprising the secretory leader sequence ("preproenzyme") (corresponding to residues 1-707 of the SEQ ID NO: 1 in the case of human MMP9), immature latent MMP9 lacking the secretory leader sequence ("proenzyme") (corresponding to residues 20-707 of SEQ ID NO: 1 in the case of human MMP9 ), I have "activated enzyme" (corresponding to residues 107-707 of SEQ ID NO: 1 in the case of human MMP9), or any fragment of MMP9.

The antibodies according to the invention, or fragments thereof, can bind to MMP9 by interacting with an epitope comprising amino acids located anywhere in the protein, e.g. eg, in the prodomain, the catalytic domain, in particular in the Fn repeats or the OG domain linker, the recognized amino acids being located at one or more sites within the protein. In a particular embodiment, the antibodies according to the invention, antigen-binding fragments thereof, bind to MMP9 by interacting with an epitope comprising amino acids located in the catalytic domain of MMP9, in particular human MMP9. In a more particular embodiment, the antibodies according to the invention, antigen-binding fragments thereof, bind to MMP9 by interacting with an epitope comprising at least one amino acid within a region consisting of SEQ ID NO: 41, at least one amino acid within a region consisting of SEQ ID NO: 42, and at least one amino acid within a region consisting of SEQ ID NO: 43, located in the catalytic domain of human MMP9. In an even more particular embodiment, the antibodies according to the invention, antigen-binding fragments thereof, bind to MMP9 by interacting with an epitope comprising the amino acids of a region consisting of SEQ ID NO: 41, the amino acids of a region consisting of SEQ ID NO: 42, and the amino acids of a region consisting of SEQ ID NO: 43, located in the catalytic domain of human MMP9.

In a more particular embodiment, the antibodies according to the invention, antigen-binding fragments thereof, bind to MMP9 by interacting with an epitope comprising at least one, at least two, at least three, at least four, or at least least five amino acids from a region consisting of SEQ ID NO: 41, at least one, at least two, at least three, at least four, or at least five amino acids from a region consisting of SEQ ID NO: 42, and at least one, at least two, at least three, at least four, or at least five amino acids from a region consisting of SEQ ID NO: 43, located in the catalytic domain of human MMP9. Thus, in one embodiment, the antibodies according to the invention, or fragments thereof, not only bind to MMP9 but also neutralize or inhibit the activity of MMP9 (eg, the catalytic activity of MMP9) by inhibiting the processing of the preproenzyme and / or proenzyme to the catalytically active enzyme and / or by inhibiting the proteolytic activity of the activated enzyme.

In a particular embodiment, the antibodies according to the invention, or fragments of these, not only bind to MMP9 but also neutralize or inhibit the activity of MMP9 (eg, the catalytic activity of MMP9) by inhibiting the proteolytic activity of activated MMP9 enzyme.

In a particular embodiment, the antibodies according to the present invention show high specificity and inhibitory activity for human MMP9 and can show cross-reactivity with Cynomologus monkey MMP9 ( Macaca fascicularis), rat MMP9 and / or mouse MP9.

In a particular embodiment, the antibodies according to the invention or fragments of these inhibit the activity of MMP9 and, as an option, also have a weak inhibitory activity, or virtually no activity. inhibitory (that is, negligible or undetectable activity) towards other matrix metalloproteinases (MMPs) such as MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP12, MMP13, MMP14, MMP16, MMP17, MMP19.

In a particular embodiment, the antibodies according to the invention, or fragments thereof, exhibit a neutralizing activity towards MMP9 and a weak or virtually no (ie negligible or undetectable) neutralizing activity towards MMP2.

The ability of an antibody to block or neutralize the activity of its protein can be assessed by its potency as defined herein, which is itself reflected, for example, by the IC ⁵⁰ value. Commonly, the neutralizing activity of an anti-MMP9 antibody can be determined by in vitro cell-free assays , or in vivo assays or in vitro functional assays such as a human cancer cell line invasion assay. In a human cancer cell line invasion assay in Transwell® culture plates, cancer cells degrade and migrate through a basement membrane matrix (Matrigel®), thereby mimicking the in vivo process of intravasation of tumor cells in nearby blood vessels and extravasation and invasion into distant tissue.

In some embodiments, the inhibitory or neutralizing potency (eg, inversely correlated to the IC ⁵⁰ value) of the antibodies, and fragments thereof, according to the invention for human MMP9 is at least 2 times, at least 5 times, at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than its neutralizing potency for non-human MMP9.

In some embodiments, the inhibitory or neutralizing potency e.g. g., inversely correlated to the IC ⁵⁰ value, of antibodies, and fragments of these, according to the invention on MMP9 is at least 10 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than its inhibitory or neutralizing potency on MMP2.

In some embodiments, the antibodies, and fragments thereof, according to the invention have an IC ⁵⁰ equal to or less than 100 nM, in particular less than 50 nM, more particularly less than 20 nM, less than 10 nM, less than 8 nM, less than 7 nM, less than 6 nM, less than 5 nM, less than 4 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.3 nM, less than 0.2 nM, or less than 0.1 nM; to inhibit the catalytic activity of MMP9 on gelatin.

The ability of an antibody to block or neutralize the activity of its target protein can also be assessed by its inhibition efficacy as defined herein, which is reflected by itself, for example, by the percentage (%) of the value. inhibition.

In some embodiments, the antibodies, and fragments thereof, according to the invention have an efficiency equal to greater than 50%, in particular equal to or greater than 60%, in particular equal to or greater than 70%, in particular equal to or greater to 80%, in particular equal to or greater than 90%, in particular equal to or greater than 95%, in particular equal to 100%, to inhibit the processing of the preproenzyme and / or proenzyme and / or inhibiting the proteolytic activity of MMP9 activated, as determined, for example, in an assay measuring the catalytic activity of MMP9 towards gelatin as described in the Examples section.

In a particular embodiment, the antibodies, and fragments thereof, according to the invention have an efficiency equal to or greater than 50%, in particular equal to or greater than 60%, in particular equal to or greater than 70%, in particular equal to or greater than 80%, in particular equal to or greater than 90%, in particular equal to or greater than 95%, in particular equal to 100%, to inhibit the proteolytic activity of activated MMP9, as determined, for example, in an assay which measures the catalytic activity of MMP9 towards gelatin as described in the Examples section.

It is understood that any variant of an antibody according to the invention, or fragment thereof, ie described herein, is capable of binding to MMP9 and optionally neutralizing the activity of MMP9. In a particular embodiment, such a variant may show the same or even higher binding affinity for MMP9 and / or the same or even higher potency and / or the same or higher species selectivity and / or the same or higher selectivity for MMP9, and / or the same or greater neutralizing efficacy, compared to the parental antibody fragment from which said variant is obtained.

Antibodies according to the invention can be monoclonal antibodies, polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, and other engineered antibodies provided that their characteristic properties of the invention are maintained, in particular the ability to bind to the target antigen, plus specifically to the same epitope of MMP9 as recognized by the antibodies of the invention, and optionally the ability to neutralize MMP9 activity.

In a particular embodiment of the invention, the MMP9-specific antibodies according to the invention, or fragments of these that specifically bind to MMP9, are monoclonal antibodies.

In another particular embodiment of the invention, the MMP9-specific antibodies according to the invention, or fragments thereof, that specifically bind to MMP9, are human antibodies.

The MMP9-specific antibodies according to the invention, or fragments thereof that specifically bind to MMP9, can be characterized by their portion that interacts with the target protein, in particular by their variable region, which commonly comprises a variable region of the heavy chain and a variable region of the light chain.

Characteristics of MMP9-binding proteins in relation to their heavy chain variable region

In one embodiment, the invention relates to isolated MMP9-specific antibodies or antigen-binding fragments thereof comprising a heavy chain variable region comprising: an amino acid sequence of SEQ ID NO: 11, or a variant of SEQ ID NO: 11, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: N53Q, N53R, N53K, N53H, D61E, S62T, M100LI, D84A and V89L. SEQ ID NO: 11, as defined above, comprises:

(i) a heavy chain CDR1 of SEQ ID NO: 2

(ii) a heavy chain CDR2 of SEQ ID NO: 3

(iii) a heavy chain CDR3 of SEQ ID NO: 4

In a particular embodiment of the invention, at least one amino acid at positions 53 and 61 of said CDR2 of the heavy chain, and / or one amino acid at position 100J of said CDR3 of the heavy chain, are replaced by a different amino acid, in particular with at least one of the following substitutions: N53Q or N53R, D61E, M100JI. In a particular embodiment of the invention, at least one amino acid at positions 53, 61 and 62 of said CDR2 of the heavy chain, and / or one amino acid at position 100L of said CDR3 of the heavy chain, are replaced by an amino acid different, in particular with at least one of the following substitutions: n53Q, N53R, N53K, N53h, D61E, S62T or M100L1.

In another particular embodiment of the invention, said CDR2 variant of the heavy chain has an amino acid sequence selected from: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and ID. SEQ NO: 9. In another particular embodiment of the invention, said CDR2 variant of the heavy chain has an amino acid sequence selected from: SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.

In another particular embodiment of the invention, said CDR3 variant of the heavy chain has an amino acid sequence SEQ ID NO: 10.

In particular, the antibodies specific for MMP9 or fragment thereof according to the invention comprise a variant of SEQ ID NO: 11, wherein at least one amino acid at positions 53 and 61 of the CDR2 of the heavy chain, wherein at least one amino acid at position 100J of the CDR3 of the heavy chain, and at positions 84 and 89 of the variable framework region of the heavy chain are replaced by a different amino acid, in particular with at least one of the following substitutions: N53Q, N53R, D61E, M100JI, D84A and V89L. In particular, the specific antibodies for MMP9 or fragments of these according to the invention comprise a variant of SEQ ID NO: 11, wherein at least one amino acid at positions 53, 61 and 62 of the CDR2 of the heavy chain, wherein at least one amino acid at position 100L of the CDR3 of the heavy chain, and at positions 84 and 89 of the variable framework region of the heavy chain are replaced by a different amino acid, in particular with at least one of the following substitutions: N53Q, N53R, N53K, N53H, D61E, S62T, D84A, V89L and M100LLI.

More particularly, the MMP9-specific antibodies or fragments thereof according to the invention comprise a variant of SEQ ID NO: 11, wherein the amino acids at positions 84 and 89 of the variable framework region of the heavy chain and at least an amino acid at positions 53 and 61 of the CDR2 of the heavy chain, and at position 100J of the CDR3 of the heavy chain are replaced by a different amino acid, in particular with D84A and V89L substitutions and at least one of the following substitutions: N53Q, N53R, D61E, and M100JI.

In particular, the specific antibodies for MMP9 or fragment thereof according to the invention comprise a variant of SEQ ID NO: 11, wherein at least one amino acid at positions 53, 61 and 62 of the CDR2 of the heavy chain, wherein at least one amino acid at position 100L of the CDR3 of the heavy chain, and at positions 30, 84 and 89 of the variable framework region of the heavy chain are replaced by a different amino acid, in particular with at least one of the following substitutions: N53Q, N53R, N53k, N53H, D61E, S62T, M100LI, D84A and V89L.

Specific examples of the heavy chain variable region comprised in antibodies or fragments thereof according to the invention include:

(i) the amino acid sequence SEQ ID NO: 11,

(ii) the amino acid sequence SEQ ID NO: 12

(iii) the amino acid sequence SEQ ID NO: 13

(iv) the amino acid sequence SEQ ID NO: 14

(v) the amino acid sequence SEQ ID NO: 15

(vi) the amino acid sequence SEQ ID NO: 16.

Alternative specific examples of the heavy chain variable region comprised in antibodies or fragments thereof, according to the invention include:

(i) the amino acid sequence SEQ ID NO: 17

(ii) the amino acid sequence SEQ ID NO: 18

(iii) the amino acid sequence SEQ ID NO: 19

(iv) the amino acid sequence SEQ ID NO: 20.

Alternative specific examples of the heavy chain variable region comprised in the antibodies or fragments thereof according to the invention include:

(i) the amino acid sequence SEQ ID NO: 48

(ii) the amino acid sequence SEQ ID NO: 53

(iii) the amino acid sequence SEQ ID NO: 54

(iv) the amino acid sequence SEQ ID NO: 55

(v) the amino acid sequence SEQ ID NO: 56.

Alternative specific examples of the heavy chain variable region comprised in antibodies or fragments thereof according to the invention include:

(i) the amino acid sequence SEQ ID NO: 12:

(ii) the amino acid sequence SEQ ID NO: 13:

(iii) the amino acid sequence SEQ ID NO: 14:

(iv) the amino acid sequence SEQ ID NO: 15:

(v) the amino acid sequence SEQ ID NO: 16:

(vi) the amino acid sequence SEQ ID NO: 17:

(vii) the amino acid sequence SEQ ID NO: 18:

(viii) the amino acid sequence SEQ ID NO: 19:

(ix) the amino acid sequence SEQ ID NO: 20: and

(x) the amino acid sequence of SEQ ID NO: 11.

Characteristics of MMP9-binding proteins in relation to their light chain variable region

In one embodiment, the invention relates to isolated antibodies specific for MMP9 antigen-binding fragments thereof comprising a heavy chain variable region as already described and further comprising a light chain variable region selected from the following group:

(1) SEQ ID NO: 24, or a variant of SEQ ID NO: 24 with the V104L substitution:

(2) SEQ ID NO: 67, or a variant of SEQ ID NO: 67, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: V46L, V71A and V104L;

(3) SEQ ID NO: 29, or a variant of SEQ ID NO: 29, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: R8A, V19I, L47M, T60N, L79Q, and A81E; and (4) SEQ ID NO: 69, or a variant of SEQ ID NO: 69, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: R8A, V19I, L47M, T60N, L79Q, A81E and F86Y. SEQ ID NO: 24 and SEQ ID NO: 67 as defined above comprise:

(i) a light chain CDR1 of SEQ ID NO: 21

(ii) a light chain CDR2 of SEQ ID NO: 22;

(iii) a light chain CDR3 of SEQ ID NO: 23.

In another embodiment, MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variant of SEQ ID NO: 67, wherein 1, 2, or 3 amino acids of at least one of CDR1, CDR2, and / or CDR3 of the light chain, and / or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids of the variable framework region of the light chain are replaced by a different amino acid.

In another particular embodiment of the invention, the MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO: 67 where 1 amino acid is substituted by a different amino acid, wherein said variable region of the light chain is SEQ ID NO: 68. In another particular embodiment of the invention, antibodies specific for MMP9, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO. : 67 where 3 amino acids are substituted by different amino acids, where said light chain variable region is SEQ ID NO: 57.

In particular, antibodies specific for MMP9 or fragment thereof according to the invention comprise a variant of SEQ ID NO: 24, wherein at least one amino acid at position 104 of the variable framework region of the light chain is replaced by a different amino acid, in particular with at least the following substitution:

V104L.

A specific example of the variable region comprised in the antibodies or fragments thereof according to the invention includes the amino acid sequence SEQ ID NO: 25.

In particular, the specific antibodies for MMP9 or fragment thereof according to the invention comprise a variant of SEQ ID NO: 67, wherein an amino acid at positions 104, of the light chain framework region is replaced by a different amino acid , in particular with at least the following substitution: V104L.

A specific example of the variable region comprised in the antibodies or fragments thereof according to the invention includes the amino acid sequence SEQ ID NO: 68.

In particular, antibodies specific for MMP9 or fragment thereof according to the invention comprise a variant of SEQ ID NO: 67, where at least three amino acids at positions 46, 71 and 104 of the light chain framework region are substituted for a different amino acid, in particular with at least the following substitution: V46L, V71A and V104L.

A specific example of the variable region comprised in antibodies or fragments thereof according to the invention includes the amino acid sequence SEQ ID NO: 57.

Another aspect of the invention provides a light chain variable region of SEQ ID NO: 29 or SEQ ID NO: 69, as defined above, comprising:

(i) a light chain CDR1 of SEQ ID NO: 26;

(ii) a light chain CDR2 of SEQ ID NO: 27;

(iii) a light chain CDR3 of SEQ ID NO: 28.

In another particular embodiment of the invention, the MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO: 69 in which 4 amino acids are substituted by different amino acids, wherein said light chain variable region is SEQ ID NO: 58.

In another particular embodiment of the invention, the MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO: 69 in which 5 amino acids are substituted by different amino acids, wherein said Light chain variable region is selected from SEQ ID NO: 59, SEQ ID NO: 60 and SEQ ID NO: 61.

In another particular embodiment of the invention, the MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO: 29 where 6 amino acids are substituted by different amino acids, wherein said light chain variable region is SEQ ID NO: 30.

In another particular embodiment of the invention, the MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO: 69 where 6 amino acids are substituted by different amino acids, wherein said Light chain variable region is selected from SEQ ID NO: 62 and SEQ ID NO: 70.

In another particular embodiment of the invention, the MMP9-specific antibodies, or antigen-binding fragments thereof, comprise a variable region of the light chain of SEQ ID NO: 69 in which 7 amino acids are substituted by different amino acids, wherein said light chain variable region is SEQ ID NO: 63.

In particular, the specific antibodies for MMP9 or fragments thereof, according to the invention comprise a variant of SEQ ID NO: 29, where at least one amino acid at positions 8, 19, 47, 60, 79 and 81 of the variable framework region of the light chain is replaced by a different amino acid, in particular with at least one of the following substitutions: R8A, V19I, L47M, T60N, L79Q, and A81E.

A specific example of the variable region comprised in antibodies or fragments thereof according to the invention includes the amino acid sequence SEQ ID NO: 30.

In particular, the MMP9-specific antibodies or fragments thereof according to the invention comprise a variant of SEQ ID NO: 69, wherein at least one amino acid at positions 8, 19, 47, 60, 79, 81, 86 of the variable framework region of the light chain are replaced by a different amino acid, in particular with at least one of the following substitutions: R8A, V19I, L47M, ^t 60N, L79Q, A81E and F86Y.

A specific example of the variable region comprised in the antibodies or fragments thereof according to the invention includes the amino acid sequence SEQ ID NO: 70. Specific examples of the variable region of the light chain comprised in the antibodies or fragments thereof according to the invention include:

(i) the amino acid sequence ID s Ec NO: 24,

(ii) the amino acid sequence SEQ ID NO: 25,

(iii) the amino acid sequence SEQ ID NO: 29,

(iv) the amino acid sequence SEQ ID NO: 30,

(v) the amino acid sequence SEQ ID NO: 57,

(vi) the amino acid sequence SEQ ID NO: 58,

(vii) the amino acid sequence SEQ ID NO: 59,

(viii) the amino acid sequence SEQ ID NO: 60,

(ix) the amino acid sequence SEQ ID NO: 61,

(x) the amino acid sequence SEQ ID NO: 62,

(xi) the amino acid sequence SEQ ID NO: 63,

(xii) the amino acid sequence SEQ ID NO: 67,

(xiii) the amino acid sequence SEQ ID NO: 68,

(xiv) the amino acid sequence SEQ ID NO: 69,

(xv) the amino acid sequence SEQ ID NO: 70.

Other specific examples of the variable region of the light chain comprised in the antibodies or fragments thereof according to the invention include:

(i) the amino acid sequence of SEQ ID NO: 67:

(ii) the amino acid sequence of SEQ ID NO: 68:

(iii) the amino acid sequence of SEQ ID NO: 69: and

(iv) the amino acid sequence of SEQ ID NO: 70.

A part corresponding to a constant region of an antibody is optionally comprised in the isolated MMP9-specific antibodies, or antigen-binding fragments thereof, according to the invention.

Depending on the proposed function of the antibodies and, in particular, the effector functions that may be necessary, a constant region of an antibody may or may not be present within the antibodies according to the invention.

Commonly, when present within antibodies or antigen-binding fragments thereof, according to the invention, the heavy chain constant region or portion thereof may be of any antibody isotype. For example, the heavy chain constant region or portion thereof can be that of an antibody selected from IgG (eg, IgG1, IgG2, IgG3, IgG4), IgA (eg, IgA1, IgA2), IgD, IgE, IgM (eg, IgM1, IgM2). It can be, in particular, the constant region or portion thereof of an IgG, more particularly IgG4.

In particular, the constant region domains of human IgG can be used, especially of the IgG1 and IgG3 isotypes when the antibody molecule is intended for therapeutic uses and effector functions of antibodies are required. Otherwise, the IgG2 and IgG4 isotypes can be used when the antibody molecule is intended for therapeutic purposes and effector functions of antibodies are not required, e.g. eg, to simply block MMP9 activity.

It will be appreciated that sequence variants of these constant region domains can also be used. For example, the heavy chain constant region or portion thereof may be that of a manipulated variant of IgG4, such as an IgG4 variant comprising S228P, R409K and a terminal lysine deletion, corresponding to the amino acid sequence SEQ ID NO: 40. When antigen-binding fragments thereof are present within antibodies, according to the invention, the light chain constant region or portion thereof may be from any light chain constant region. For example, the light chain constant region or portion thereof can be from the kappa or lambda light chain.

In a particular aspect of the invention, MMP9-specific antibodies, or antigen-binding fragments thereof, comprise: (i) at least one heavy chain comprising a variable region as described herein and a constant region or portion thereof from an IgG antibody, and (ii) at least one light chain comprising a variable region as described herein and a constant region or portion thereof of a lambda light chain (in particular lambda 2).

In a particular embodiment, the constant region or portion thereof of the heavy chain and / or the light chain, which is comprised in the antibodies according to the invention, has an amino acid sequence that has been modified compared to its sequence of original amino acids, according to the methods known in the art, to increase the chemical stability of the antibodies, decrease their aggregation, increase their production, in particular in antibody-producing cells (eg, HEK293 cells, CHO cells), and / or eliminate their ability to exchange semi-molecules that could effectively result in monovalent antibodies.

Examples of amino acids within the amino acid sequence of the constant region of an antibody that affect the stability of the antibody include the mutation of amino acid S228P (EU numbering) in the heavy chain of human IgG4 that stabilizes the hinge domain of the antibody ( Angal et al., 1993, Molec. Immunol. 30: 105-108) and avoids Fc-Fc interactions ( Rispens et al., 2013, Mol. Immunol. 53: 35-42), a Lys (K) instead of a Arg (R) at allotypic position 409 (EU numbering) which increases the strength of the CH3-CH3 interaction in IgG4 ( Allberse et al, 2002, Immunology 105: 9-19). In addition, in order to simplify monitoring of monoclonal antibody loading heterogeneity, the C-terminal lysine can be removed from the human IgG4 heavy chain. In another embodiment, MMP9-specific antibodies, or antigen-binding fragments thereof, in accordance with the invention comprise at least one heavy chain comprising a variable region as described herein and a constant region or portion thereof of an IgG4 antibody, wherein the amino acid sequence of the IgG4 constant region comprises the following amino acid modifications: S228P (EU numbering), R409K (EU numbering), terminal Lys deletion (K), wherein said modified constant region it is represented by SEQ ID ⁿ O: 40.

In another embodiment, MMP9-specific antibodies, or antigen-binding fragments thereof, in accordance with the invention comprise at least one light chain comprising a variable region as described herein and a constant region or portion thereof of an IgG4 antibody, wherein said constant region is represented by SEQ ID NO: 66.

In another particular embodiment of the invention, said CDR2 variant of the heavy chain has an amino acid sequence selected from: SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. In another particular embodiment of the invention, said CDR2 variant of the heavy chain has an amino acid sequence selected from: SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51.

In another particular embodiment of the invention, said CDR3 variant of the heavy chain has an amino acid sequence SEQ ID NO: 10.

In another particular embodiment of the invention, said CDR3 variant of the heavy chain has an amino acid sequence SEQ ID NO: 52.

The antibodies of the invention have at least one antigen-binding site, e.g. eg, one or two antigen-binding sites. In certain embodiments, for example, for peptides comprising ^c D ^r , a variable domain may contain only the CDRs linked through short linker peptides rather than full framework regions.

In some embodiments, the isolated antibodies and antigen-binding fragments thereof, according to the invention, are glycosylated. Commonly, monosaccharides like N-acetylglucosamine, mannose, glucose, galactose, fucose, sialic acid, etc., are assembled to oligosaccharides at individual glycosylation sites on the antibody.

In another particular embodiment, the invention provides an isolated antibody specific for MMP9, or an antigen-binding fragment thereof, wherein said antibody or fragment thereof comprises:

(i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, or a variant of SEQ ID NO: 11, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 11: N ⁵⁴ Q, N ⁵⁴ R, N ⁵⁴ K, N ⁵⁴ H, D ⁶² E, S ⁶³ T, M ¹¹⁶ I, D ^as A and V ⁹³ L and

(ii) further comprising a light chain variable region selected from the following group:

(1) SEQ ID NO: 24, or a variant of SEQ ID NO: 24, wherein the amino acid at position 107 of SEQ ID NO: 24 is substituted with the substitution: V ¹⁰⁷ L;

(2) SEQ ID NO: 67, or a variant of SEQ ID NO: 67, wherein at least one amino acid is substituted by a different amino acid selected from the following substitution in the sequence of SEQ ID NO: 67: V ⁴⁵ L, V ⁷⁰ A and V ¹⁰⁷ L;

(3) SEQ ID NO: 29, or a variant of SEQ ID NO: 29, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 29: R ^a A, V ^{1 a} I, L ⁴⁹ M, T ⁶² N, L ^a1 Q, and A ^a3 E; Y

(4) SEQ ID NO: 69, or a variant of SEQ ID NO: 69, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 69: R ^a A, V ^{1 a} I, L ⁴⁹ M, T ⁶² N, L ^a1 Q, A ^a3 E and F ^aa Y.

In another particular embodiment, the invention provides an isolated antibody specific for MMP9, or an antigen-binding fragment thereof, wherein a variable region of the heavy chain comprises an amino acid sequence selected from:

(i) the amino acid sequence SEQ ID NO: 12:

(ii) the amino acid sequence SEQ ID NO: 13:

(iii) the amino acid sequence SEQ ID NO: 14:

(iv) the amino acid sequence SEQ ID NO: 15:

(v) the amino acid sequence SEQ ID NO: 16:

(vi) the amino acid sequence SEQ ID NO: 17:

(vii) the amino acid sequence SEQ ID NO: 18:

(viii) the amino acid sequence SEQ ID NO: 19:

(ix) the amino acid sequence SEQ ID NO: 20: and

(x) the amino acid sequence SEQ ID NO: 11, and wherein a variable region of the light chain comprises an amino acid sequence selected from:

(xi) the amino acid sequence SEQ ID NO: 24;

(xii) the amino acid sequence SEQ ID NO: 25;

(xiii) the amino acid sequence SEQ ID NO: 29;

(xiv) the amino acid sequence SEQ ID NO: 30;

(xv) the amino acid sequence SEQ ID NO: 57;

(xvi) the amino acid sequence SEQ ID NO: 58;

(xvii) the amino acid sequence SEQ ID NO: 59;

(xviii) the amino acid sequence SEQ ID NO: 60:

(xix) the amino acid sequence SEQ ID NO: 61:

(xx) the amino acid sequence SEQ ID NO: 62:

(xxi) the amino acid sequence SEQ ID NO: 63:

(xxii) the amino acid sequence SEQ ID NO: 67:

(xxiii) the amino acid sequence SEQ ID NO: 68:

(xxiv) the amino acid sequence SEQ ID NO: 69: and

(xxv) the amino acid sequence SEQ ID NO: 70.

In another particular embodiment, the invention provides an isolated antibody specific for MMP9, or an antigen-binding fragment thereof, wherein a variable region of the heavy chain comprises an amino acid sequence with (a), and a variable region of the light chain comprises an amino acid sequence with (b) selected from combinations of the group consisting of:

(b): the amino acid sequence SEQ ID NO: 24, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16. 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 24, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 25, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 25, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 29, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15. 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 29, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 30, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 30, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 57, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 57, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 58, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 58, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 59, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 59, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 60, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15. 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 60, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53. 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 61, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 61, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 62, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15. 16, 17, 18. 19 and 20:

(b): the amino acid sequence SEQ ID NO: 62, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 63, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 63, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 67, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 67, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 68, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15, 16, 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 68, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 69, and (a): the selected amino acid sequence of SEQ ID NO: 11. 12, 13, 14, 15. 16. 17, 18, 19 and 20:

(b): the amino acid sequence SEQ ID NO: 69, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56:

(b): the amino acid sequence SEQ ID NO: 70, and (a): the amino acid sequence selected from SEQ ID NO: 11. 12, 13, 14, 15. 16, 17, 18, 19 and 20: Y

(b): the amino acid sequence SEQ ID NO: 70, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56.

As will be understood by a person skilled in the art, one aspect of the present invention relates to isolated MMP9-specific antibodies or antigen-binding fragments thereof, which are characterized by some of the characteristics described herein without necessarily understanding all of the aforementioned. features.

For example, in one aspect, isolated MMP9-specific antibodies or antigen-binding fragments thereof are provided that are characterized by any of the characteristics described herein with respect to the sequences of their variable regions and / or constant regions.

In a particular embodiment of said aspect, said antibodies or fragments thereof are further characterized by their binding to MMP9 interacting with an epitope on MMP9 as described herein, in particular an epitope that comprises at least one amino acid within a region that consists of SEQ ID NO: 41, at least one amino acid within a region consisting of SEQ ID NO: 42, and at least one amino acid within a region consisting of SEQ ID NO: 43, located in the domain catalyst of human MMP9.

In an alternative aspect, isolated MMP9-specific antibodies or antigen-binding fragments thereof are provided that are characterized by their binding to MMP9 by interacting with an epitope on MMP9 as described herein, in particular an epitope comprising al least one amino acid within a region consisting of SEQ ID NO: 41, at least one amino acid within a region consisting of SEQ ID NO: 42, and at least one amino acid within a region consisting of SEQ ID NO: 43, located in the catalytic domain of human MMP9.

In a particular embodiment of said alternative aspect, said antibodies or fragments thereof are further characterized by any of the characteristics described herein with respect to the sequences of their variable regions and / or constant regions.

Examples of MMP9-specific antibodies or antigen-binding fragment thereof

Examples of antibodies and fragments thereof according to the invention include those comprising the variable domains listed in Table 2 (see also Figures 3, 4, 5).

Table 2

Thus, in a specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 11

(ii) the amino acid sequence of SEQ ID NO: 12

(iii) the amino acid sequence of SEQ ID NO: 13

(iv) the amino acid sequence of SEQ ID NO: 14

(v) the amino acid sequence of SEQ ID NO: 15

(vi) the amino acid sequence of SEQ ID NO: 16

(vii) the amino acid sequence of SEQ ID NO: 17

(viii) the amino acid sequence of SEQ ID NO: 18

(ix) the amino acid sequence of SEQ ID NO: 19

(x) the amino acid sequence of SEQ ID NO: 20

(xi) the amino acid sequence of SEQ ID NO: 48

(xii) the amino acid sequence of SEQ ID NO: 53

(xiii) the amino acid sequence of SEQ ID NO: 54

(xiv) the amino acid sequence of SEQ ID NO: 55

(xv) the amino acid sequence of SEQ ID NO: 56, and

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 24

(ii) the amino acid sequence of SEQ ID NO: 25

(iii) the amino acid sequence of SEQ ID NO: 29

(iv) the amino acid sequence of SEQ ID NO: 30

(v) the amino acid sequence of SEQ ID NO: 57

(vi) the amino acid sequence of SEQ ID NO: 58

(vii) the amino acid sequence of SEQ ID NO: 59

(viii) the amino acid sequence of SEQ ID NO: 60

(ix) the amino acid sequence of SEQ ID NO: 61

(x) the amino acid sequence of SEQ ID NO: 62

(xi) the amino acid sequence of SEQ ID NO: 63

(xii) the amino acid sequence of SEQ ID NO: 67

(xiii) the amino acid sequence of SEQ ID NO: 68

(xiv) the amino acid sequence of SEQ ID NO: 69

(xv) the amino acid sequence of SEQ ID NO: 70.

Thus, in a specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 11

(ii) the amino acid sequence of SEQ ID NO: 12

(iii) the amino acid sequence of SEQ ID NO: 13

(iv) the amino acid sequence of SEQ ID NO: 14

(v) the amino acid sequence of SEQ ID NO: 15

(vi) the amino acid sequence of SEQ ID NO: 16

(vii) the amino acid sequence of SEQ ID NO: 17

(viii) the amino acid sequence of SEQ ID NO: 18

(ix) the amino acid sequence of SEQ ID NO: 19

(x) the amino acid sequence of SEQ ID NO: 20

(xi) the amino acid sequence of SEQ ID NO: 48

(xii) the amino acid sequence of SEQ ID NO: 53

(xiii) the amino acid sequence of SEQ ID NO: 54

(xiv) the amino acid sequence of SEQ ID NO: 55

(xv) the amino acid sequence of SEQ ID NO: 56, and

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 57

(ii) the amino acid sequence of SEQ ID NO: 58

(iii) the amino acid sequence of SEQ ID NO: 59

(iv) the amino acid sequence of SEQ ID NO: 60

(v) the amino acid sequence of SEQ ID NO: 61

(vi) the amino acid sequence of SEQ ID NO: 62

(vii) the amino acid sequence of SEQ ID NO: 63

(viii) the amino acid sequence of SEQ ID NO: 67

(ix) the amino acid sequence of SEQ ID NO: 68

(x) the amino acid sequence of SEQ ID NO: 69

(xi) the amino acid sequence of SEQ ID NO: 70.

Thus, in a specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 12

(ii) the amino acid sequence of SEQ ID NO: 17

(iii) the amino acid sequence of SEQ ID NO: 18

(iv) the amino acid sequence of SEQ ID NO: 19

(v) the amino acid sequence of SEQ ID NO: 20, and

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 68

(ii) the amino acid sequence of SEQ ID NO: 70.

Thus, in a specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 12

(ii) the amino acid sequence of SEQ ID NO: 18

(iii) the amino acid sequence of SEQ ID NO: 20, and

(2) an amino acid sequence of the light chain variable region of SEQ ID NO: 68.

Thus, in a specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 12

(ii) the amino acid sequence of SEQ ID NO: 17

(iii) the amino acid sequence of SEQ ID NO: 18

(iv) the amino acid sequence of SEQ ID NO: 19

(v) the amino acid sequence of SEQ ID NO: 20, and

(2) an amino acid sequence of the light chain variable region of SEQ ID NO: 70.

Thus, in a specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 11

(ü) the amino acid sequence of SEQ ID NO: 12

(iii) the amino acid sequence of SEQ ID NO: 13

(iv) the amino acid sequence of SEQ ID NO: 14

(v) the amino acid sequence of SEQ ID NO: 15

(vi) the amino acid sequence of SEQ ID NO: 16

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 24

(ii) the amino acid sequence of SEQ ID NO: 25.

In another specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 12

(ii) the amino acid sequence of SEQ ID NO: 17

(iii) the amino acid sequence of SEQ ID NO: 18

(iv) the amino acid sequence of SEQ ID NO: 19

(v) the amino acid sequence of SEQ ID NO: 20, and

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 24

(ii) the amino acid sequence of SEQ ID NO: 25.

More particularly, isolated MMP9-specific antibodies or antigen-binding fragments thereof, according to the invention, may comprise:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 18

(ii) the amino acid sequence of SEQ ID NO: 20, and

(2) an amino acid sequence of the light chain variable region of SEQ ID NO: 25.

In another particular embodiment, the MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 11

(ii) the amino acid sequence of SEQ ID NO: 12

(iii) the amino acid sequence of SEQ ID NO: 13

(iv) the amino acid sequence of SEQ ID NO: 14

(v) the amino acid sequence of SEQ ID NO: 15

(vi) the amino acid sequence of SEQ ID NO: 16, and

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 29

(ii) the amino acid sequence of SEQ ID NO: 30

In another specific embodiment, MMP9-binding proteins according to the invention include isolated MMP9-specific antibodies or antigen-binding fragments thereof, comprising:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 12

(ii) the amino acid sequence of SEQ ID NO: 17

(iii) the amino acid sequence of SEQ ID NO: 18

(iv) the amino acid sequence of SEQ ID NO: 19

(v) the amino acid sequence of SEQ ID NO: 20, and

(2) a variable region of the light chain selected from:

(i) the amino acid sequence of SEQ ID NO: 29

(ii) the amino acid sequence of SEQ ID NO: 30.

More particularly, isolated MMP9-specific antibodies or antigen-binding fragments thereof according to the invention may comprise:

(1) a heavy chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 17

(ii) the amino acid sequence of SEQ ID NO: 18

(iii) the amino acid sequence of SEQ ID NO: 19

(iv) the amino acid sequence of SEQ ID NO: 20, and

(2) an amino acid sequence of the light chain variable region of SEQ ID NO: 30.

More particularly, isolated MMP9-specific antibodies or antigen-binding fragments thereof according to the invention may comprise a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO. : 70.

Conjugates comprising a helper molecule

In another aspect of the invention, the isolated antibodies or antigen-binding fragment thereof, according to the invention, are optionally conjugated to an accessory molecule, and are also referred to herein as "conjugated antibodies" or "antibody fragments. conjugated ".

The accessory molecule can be conjugated to the antibody or antibody fragment directly or through a spacer of suitable length, for example, as described in Kellogg et al., 2011, Bioconjug Chem, 22: 717 27).

In one embodiment, particularly adapted for therapeutic purposes, the accessory molecule can be a therapeutic effect group such as a cytotoxic agent (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragment thereof) , cytostatic, or immunomodulatory, including radioactive groups (ie, groups comprising a radionuclide or radioisotope), or small molecules.

In another embodiment, the accessory molecule comprises an antigen-binding fragment of an antibody, which, when conjugated to the antibody or antibody fragment according to the invention, forms a bispecific antibody. In particular, said bispecific antibody can be directed to two different MMPs or to two different epitopes of an MMP, such as two different epitopes of MMP9.

In a specific embodiment, the accessory molecule can be, for example, an active agent for the treatment of a disease, such as, for the treatment of Crohn's disease: the variable region of an antibody anti-TNFa, which, when conjugated to an antibody or antigen-binding fragment of the invention can form bi-specific anti-TNFa / MMP9 antibodies for the treatment of individuals suffering from moderate to severe Crohn's disease.

Conjugated antibodies and conjugated antibody fragments according to the invention can direct the drug in vivo to a site of disease (eg, a site of inflammation or a tumor) such that the conjugated helper molecule can have a therapeutic effect at the site of the disease.

In an alternative embodiment, particularly adapted for diagnostic purposes, the accessory molecule can be, for example, a tagging group that includes radioisotopes (eg, 3H, 14C, 32P, 35S, 1251), chromogenic tags, e.g. g., enzymes that can be used to convert a substrate to a color-detectable compound (eg, horseradish peroxidase, alkaline phosphatase, p-galactosidase) or fluorescent (eg, green fluorescent protein, red fluorescent protein) , spectroscopic labels (eg, fluorescent labels such as fluorescein and its derivatives such as FlTc, Texas red, cyanine dyes, fotocyan (sic), rhodamine, or labels that present a visible color), luminescent labels that include luciferins, affinity tags that can be developed by another compound specific for the tag and allowing easy detection and quantification, or any other tag used in normal ELISA.

Nucleic acids encoding the polypeptides of the invention

According to another embodiment, an isolated nucleic acid molecule is provided which encodes an antibody or antigen-binding fragment thereof according to the invention. The isolated nucleic acid according to the invention can be, for example, natural DNA or RNA or a recombinant or synthetic DNA, RNA or LNA or a recombinant nucleic acid molecule comprising any of the nucleic acid molecules according to the invention either alone or in combination. In a particular embodiment, the nucleic acid molecules according to the invention are cDNA.

In a particular embodiment, an isolated nucleic acid is provided comprising one or more of:

(1) a nucleic acid sequence encoding a heavy chain CDR1 of SEQ ID NO: 2, a heavy chain CDR2 of SEQ ID NO: 3, a heavy chain CDR3 of SEQ ID NO: 4, or a variant thereof, wherein 1, 2, or 3 amino acids of said heavy chain CDR are replaced by a different amino acid, and

(2) (i) a nucleic acid encoding a light chain CDR1 of SEQ ID NO: 21, a light chain CDR2 of SEQ ID NO: 22, a light chain CDR3 of SEQ ID NO: 23, or a variant thereof, wherein 1, 2 or 3 amino acids of said light chain CDR are replaced by a different amino acid, or

(2) (ii) a nucleic acid encoding a CDR1 of the light chain of SEQ ID NO: 26, a CDR2 of the light chain of ⁱ D SEQ NO: 27, a CDR3 of the light chain of SEQ ID NO: 28 , or a variant thereof, wherein 1, 2 or 3 amino acids of said light chain CDR are replaced by a different amino acid.

In a particular embodiment, an isolated nucleic acid is provided comprising one or more of:

(1) a nucleic acid sequence encoding a heavy chain variable region selected from:

(1) the amino acid sequence of SEQ ID NO: 11

(i the amino acid sequence of SEQ ID NO: 12

(iii) the amino acid sequence of SEQ ID NO: 13

(iv) the amino acid sequence of SEQ ID NO: 14

(v) the amino acid sequence of SEQ ID NO: 15

(vi) the amino acid sequence of SEQ ID NO: 16

(vii) the amino acid sequence of SEQ ID NO: 17

(viii) the amino acid sequence of SEQ ID NO: 18

(ix) the amino acid sequence of SEQ ID NO: 19

(x) the amino acid sequence of SEQ ID NO: 20

(xi) the amino acid sequence of SEQ ID NO: 48

(xii) the amino acid sequence of SEQ ID NO: 53

(xiii) the amino acid sequence of SEQ ID NO: 54

(xiv) the amino acid sequence of SEQ ID NO: 55

(XV) the amino acid sequence of SEQ ID NO: 56

(2) a nucleic acid sequence encoding a light chain variable region selected from:

(i) the amino acid sequence of SEQ ID NO: 24

(ii) the amino acid sequence of SEQ ID NO: 25

(iii) the amino acid sequence of SEQ ID NO: 29

(iv) the amino acid sequence of SEQ ID NO: 30

(v) the amino acid sequence of SEQ ID NO: 57

(vi) the amino acid sequence of SEQ ID NO: 58

(vii) the amino acid sequence of SEQ ID NO: 59

(viii) the amino acid sequence of SEQ ID NO: 60

(ix) the amino acid sequence of SEQ ID NO: 61

(x) the amino acid sequence of SEQ ID NO: 62

(xi) the amino acid sequence of SEQ ID NO: 63

(xii) the amino acid sequence of SEQ ID NO: 67

(xiii) the amino acid sequence of SEQ ID NO: 68

(xiv) the amino acid sequence of SEQ ID NO: 69

(xv) the amino acid sequence of SEQ ID NO: 70.

In a particular embodiment, an isolated nucleic acid is provided comprising one or more of:

(1) a nucleic acid sequence encoding a heavy chain variable region selected from:

(i) the amino acid sequence SEQ ID NO: 11,

(ii) the amino acid sequence SEQ ID NO: 12

(iii) the amino acid sequence SEQ ID NO: 13

(iv) the amino acid sequence SEQ ID NO: 14

(v) the amino acid sequence SEQ ID NO: 15

(vi) the amino acid sequence SEQ ID NO: 16, and

(2) a nucleic acid sequence encoding a light chain variable region selected from:

(i) the amino acid sequence SEQ ID NO: 24

(ii) the amino acid sequence SEQ ID NO: 25.

In a particular alternative embodiment, the invention provides an isolated nucleic acid comprising one or more of:

(1) a nucleic acid sequence encoding a heavy chain variable region selected from:

(i) the amino acid sequence SEQ ID NO: 12

(ii) the amino acid sequence SEQ ID NO: 17

(iii) the amino acid sequence SEQ ID NO: 18

(iv) the amino acid sequence SEQ ID NO: 19

(v) the amino acid sequence SEQ ID NO: 20, and

(2) a nucleic acid sequence encoding a light chain variable region of the amino acid sequence SEQ ID NO: 25.

In another particular embodiment, an isolated nucleic acid is provided comprising one or more of:

(1) a nucleic acid sequence encoding a heavy chain variable region selected from:

(i) the amino acid sequence SEQ ID NO: 11

(ii) the amino acid sequence SEQ ID NO: 12

(iii) the amino acid sequence SEQ ID NO: 13

(iv) the amino acid sequence SEQ ID NO: 14

(v) the amino acid sequence SEQ ID NO: 15

(vi) the amino acid sequence SEQ ID NO: 16, and

(2) a nucleic acid sequence encoding a light chain variable region selected from:

(i) the amino acid sequence SEQ ID NO: 29

(ii) the amino acid sequence SEQ ID NO: 30.

In a particular alternative embodiment, an isolated nucleic acid is provided comprising one or more of:

(1) a nucleic acid sequence encoding a heavy chain variable region selected from:

(i) the amino acid sequence SEQ ID NO: 12

(ii) the amino acid sequence SEQ ID NO: 17

(iii) the amino acid sequence SEQ ID NO: 18

(iv) the amino acid sequence SEQ ID NO: 19

(v) the amino acid sequence SEQ ID NO: 20, and

(2) a nucleic acid sequence encoding a light chain variable region of the amino acid sequence SEQ ID NO: 30.

In another particular embodiment, an isolated nucleic acid is provided comprising one or more of:

(1) a nucleic acid sequence comprising SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, or a variant thereof having at least 80%, at least 90%, at least 95% , at least 96%, at least 97%, at least 98%, or at least 99% identity with one of said sequences, and / or

(2) a nucleic acid sequence selected from:

a) a nucleic acid sequence comprising SEQ ID NO: 34, SEQ ID NO: 35 and SEQ ID NO: 36, or a variant thereof having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of said sequences, b) a nucleic acid sequence comprising SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, or a variant thereof that has at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of said sequences.

Vectors and host cells for the production and purification of the polypeptides of the invention

In one embodiment, the invention provides a recombinant expression vector comprising a nucleic acid molecule according to the invention, wherein the vector optionally comprises an expression control sequence, allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide. , operably linked to said nucleic acid molecule.

Numerous expression systems can be used, including, without limitation, chromosomes, episomes, and derived viruses. More particularly, the recombinant vectors used can be obtained from bacterial plasmids, transposons, yeast episomes, insert elements, yeast chromosome elements, viruses such as baculovirus, papilloma virus such as SV40, vaccinia virus, adenovirus, smallpox virus. , pseudorabies virus, retrovirus. These recombinant vectors can also be cosmids or phagemid derivatives. The nucleic acid sequence can be inserted into the recombinant expression vector by means well known to a person skilled in the art, such as, for example, those described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al, 4th Ed ., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001. The recombinant vector may include nucleotide sequences that allow, control or regulate the expression and transcription of a polynucleotide of the invention as well as the translation of a polypeptide of the invention, these sequences being selected according to the host cells that are used.

Thus, for example, an appropriate secretion signal can be integrated into the recombinant vector so that the polypeptide, encoded by the nucleic acid molecule of the invention, will be directed towards the lumen of the endoplasmic reticulum, towards the periplasmic space, over the membrane or into the extracellular medium. Choosing an appropriate secretion signal can facilitate subsequent protein purification. In another embodiment, a host cell is provided comprising a recombinant vector according to the invention.

The introduction of the recombinant vector into a host cell can be carried out according to methods that are well known to a person skilled in the art, such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis et al, 2nd ed. , McGraw-Hill Professional Publishing, 1995, and MOLECULAR CLONING: A LABORATORY MANUAL, supra, such as calcium phosphate transfection, DEAE dextran transfection, transfection, microinjection, cationic lipid transfection, electroporation, transduction, or infection.

The host cell can be, for example, bacterial cells such as E. coli or Streptomyces, cells of fungi such as Aspergillus and yeasts such as Saccharomyces, insect cells, Chinese hamster ovary (CHO) cells, C127 mouse cell line, line BHK cell from Syrian hamster cells, human embryonic kidney 293 cells (HEK 293). In a particular embodiment, the host cell is a CHO cell or a HEK 293 cell.

Host cells can be used, for example, to express a polypeptide of the invention. After purification by normal methods, the polypeptide of the invention can be used in a method described below.

For example, when using expression systems that secrete the recombinant protein, the culture medium can first be concentrated using a commercially available protein concentration filter, eg, an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration matrix. Otherwise, an anion and / or affinity exchange resin may be employed. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. Alternatively, a cation exchange step can be employed. Finally, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media can be employed to further purify the antibodies or fragments thereof. Some or all of the above purification steps, in various combinations, are well known and can be employed to provide a highly homogeneous recombinant protein.

The recombinant protein produced in bacterial culture can be isolated by initial disruption of host cells, centrifugation, extracted from cell pellets if it is an insoluble polypeptide, or from supernatant fluid if it is a soluble polypeptide, followed by one or more concentration steps. , desalting, ion exchange, affinity purification or size exclusion chromatography. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysis agents.

In another embodiment, the host cell invention can be used to produce cells capable of expressing a polypeptide according to the invention, comprising cells genetically engineered with a vector or nucleic acid according to the invention.

In another embodiment, the nucleic acid of the invention can be used in a process for producing antibodies or fragments thereof according to the invention, which comprises culturing a transformed host cell with an expression vector comprising a nucleic sequence encoding said antibodies or fragments of these under conditions sufficient to promote the expression of said polypeptides. The antibody or its fragment according to the invention is then recovered from the culture medium or cell extracts, depending on the expression system used. As is known to one skilled in the art, the procedures for the purification of a recombinant protein will vary according to such factors as the type of host cells used and whether or not the recombinant protein is secreted in the culture medium as already described. .

Compositions

The invention provides pharmaceutical or therapeutic agents as compositions and methods for treating a patient, preferably a mammalian patient, and more preferably a human patient suffering from a medical disorder, and in particular an inflammatory and / or autoimmune disease or cancer. Alternatively, the invention provides methods for preventing a medical disorder, and in particular an inflammatory and / or autoimmune disease or cancer.

In one embodiment, a pharmaceutical composition is provided comprising one or more of: (i) an MMP9-specific antibody or antigen-binding fragment thereof according to the invention, (ii) a nucleic acid according to the invention, (iii) a vector according to the invention, and / or (iv) a host cell according to the invention, and at least one pharmaceutically acceptable carrier.

The pharmaceutical compositions of the invention may contain one or more MMP9-specific antibodies or antigen-binding fragment thereof in any form described herein. The compositions of this invention may further comprise one or more additional acceptable ingredient (s) such as alum, stabilizers, antimicrobial agents, buffers, coloring agents, flavoring agents, adjuvants, and the like.

The compounds of the invention, together with a conventionally used adjuvant, carrier, diluent or excipient can be placed in the form of pharmaceutical compositions and unit dosages thereof, and in such form they can be used as solids, such as filled tablets or capsules, lyophilized or liquid forms such as solutions, suspensions, emulsions, elixirs or capsules filled with the same, all for oral use, or in the form of sterile injectable solutions for parenteral use (including subcutaneous). Such pharmaceutical compositions and dosage unit forms thereof may comprise ingredients in conventional proportions, with or without additional compounds or active ingredients, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the proposed daily dosage range that is going to be used.

The compositions of this invention can be liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. Liquid forms suitable for oral administration may include a suitable aqueous or nonaqueous vehicle with buffers, suspending and dispensing agents, colorants, flavors, and the like. The compositions can also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives. The suspending agent includes, but is not limited to, sorbitol syrup, methyl cellulose, glucose / sugar syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and hydrogenated edible fats. Emulsifying agents include, but are not limited to, lecithin, sorbitan monooleate, and acacia. Non-aqueous vehicles include, but are not limited to, oils edibles, almond oil, fractionated coconut oil, oily esters, propylene glycol and ethyl alcohol. Preservatives include, but are not limited to, methyl or propyl p-hydroxybenzoate and sorbic acid. Other materials, as well as processing techniques, and the like, are set forth in Part 5 of Remington's The Science and Practice of Pharmacy, 22nd Edition, 2012, Pharmaceutical Press and the University of the Sciences, Philadelphia College of Pharmacy.

The solid compositions of this invention may be in the form of tablets or lozenges formulated in a traditional manner. For example, tablets and capsules for oral administration may contain traditional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants, and wetting agents. Binding agents include, but are not limited to, syrup, acacia, gelatin, sorbitol, tragacanth, starch mucilage, and polyvinylpyrrolidone. Fillers include, but are not limited to, lactose, sugar, microcrystalline cellulose, cornstarch, calcium phosphate, and sorbitol. Lubricants include, but are not limited to, magnesium stearate, stearic acid, talc, polyethylene glycol, and silica. Disintegrants include, but are not limited to, potato starch and sodium starch glycolate. Wetting agents include, but are not limited to, sodium lauryl sulfate. Tablets can be coated according to methods known in the art.

Injectable compositions are commonly based on injectable sterile saline or phosphate buffered saline or other injectable carriers known in the art.

The compositions of this invention can also be formulated as transdermal formulations comprising aqueous or non-aqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch or membrane.

The compositions of this invention can also be formulated for parenteral administration including, but not limited to, continuous injection or infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents including, but not limited to, suspending, stabilizing, and dispersing agents. The composition can also be provided in a powder form for constitution with a suitable vehicle including, but not limited to, sterile, pyrogen-free water.

The compositions of this invention can also be formulated as a depot preparation, which can be administered by implantation or by intramuscular injection. The compositions can be formulated with suitable polymeric or hydrophobic materials (as an emulsion in an acceptable oil, for example), ion exchange resins, or as sparingly soluble derivatives (as a sparingly soluble salt, for example).

The compounds of this invention can also be administered in sustained release forms or sustained release drug delivery systems. A description of representative sustained release materials can also be found in Remington's Pharmaceutical Sciences.

Injectable formulations are particularly suitable for administering the compositions according to the invention.

In another embodiment, the invention provides an imaging composition or diagnostic composition comprising an antibody specific to MMP9 or an antigen-binding fragment thereof as described herein.

In another embodiment, the antibody of the invention provides a diagnostic composition comprising an MMP9-specific antibody or antigen-binding fragment thereof as described herein for the detection of active MMP9 in individuals.

In another embodiment, a diagnostic composition comprising an MMP9-specific antibody or antigen-binding fragment thereof as described herein may be used for detection of active MMP9 in individuals, wherein said antibody comprises a variable region of the chain heavy of SEQ ID NO: 19 and a light chain variable region of ID ^s E ^c NO: 70.

In one embodiment, the diagnostic compositions may comprise an antibody specific for MMP9 or antigen-binding fragment thereof as described herein, conjugated to a portion selected from the group consisting of a radioisotope, a biotin, an avidin, an streptavidin, a chromophore, a fluorophore, a chemiluminescent moiety, a hapten, and an enzyme.

Imaging compositions or diagnostic compositions according to the invention are useful for detecting elevated levels of MMP9 associated with inflammatory and / or autoimmune diseases or cancers.

Combination

According to the invention, an antibody specific for MMP9 or antigen-binding fragment thereof according to the invention can be administered alone or in combination with a co-agent useful in the prevention and / or treatment of an inflammatory disease and / or or autoimmune, eg, immunomodulatory drugs including biologics, small molecules, and vaccines.

Otherwise, an MMP9-specific antibody or antigen-binding fragment thereof according to the invention may be administered or in combination [sic] with a co-agent useful in the treatment of cancer, for example an anti-drug -cancerous such as cytotoxic drugs (Folfox, Xelox, Folfirinox, Folfox6, capecitabine, docetaxel (Taxotere), paclitaxel (Taxol), Nab / paclitaxel, 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), Capecitabine (Xeloda), Cytarabine (Ara-C), Floxuridine, Fludarabine, Gemcitabine (Gemzar), Gemcitabine-Cisplatin, Hydroxyurea, Methotrexate, Pemetrexed (Alimta), ixabepilone (Ixempra), vinblastine), Velistban (Onco vincristine), vinorelbine (Navelbine), Estramustine (Emcyt)), tyrosine kinase inhibitors (imatinib (Gleevec / Glivec), or gefitinib (Iressa), or erlotinib (Tarceva), Afatinib (Giotrif), Axitinib (Inlyta), Bosutinib (Bosulif), Crizotinib (Xalkori), Dasatinib (Sprycel), Lapatinib (Tyverb), Nilotinib (Tasigna), Pazopanib (Votrient), Sorafenib (Nexavar), Sunitinib (Sutent)) and therapeutic antibodies such as trastuzumab (Herceptin), Bevacizumab (Avastin), Cetuximab (Erbitux), Panitumumab (Vectibix), Pertuzumab (Perjeta), ipilimumab (Yervoy), Opdivomab (Yervoy), Opdivomab and pembrolizumab (Keytruda) or anti-CD20 rituximab antibody (Rituxan).

An antibody of the invention or fragment thereof may be administered to an individual prior to, at the same time or in sequence with other therapeutic regimens or co-agents useful in the prevention and / or treatment of an MMP9-related disease selected from the group that It consists of inflammatory and autoimmune diseases, inflammatory bowel diseases, cancers or tumors, fibrotic diseases, cardiovascular diseases, neurological disorders, eye diseases, or any other disease or disorder related to MMP9, in an effective therapeutic amount. The MMP9-specific antibody or antigen-binding fragment thereof according to the invention which is administered at the same time with said co-agents can be administered in the same or different composition and in the same or different routes of administration. In a particular embodiment, an antibody specific for MMP9 or antigen-binding fragment thereof according to the invention can be administered in combination with an anti-TNFa antibody for the treatment of individuals suffering from moderate to severe Crohn's disease.

Administration mode

The compositions of this invention may be administered in any form including, but not limited to, oral, parenteral, sublingual, transdermal, rectal, transmucosal, topical, via inhalation, via buccal or intranasal or intravesical administration, or combinations of these.

Parenteral administration includes, but is not limited to, intravenous, intra-arterial, intraperitoneal, subcutaneous, intramuscular, intra-thecal, and intra-articular. The compositions of this invention can also be administered in the form of an implant, which allows for slow release of the compositions as well as an i.v. infusion. slow, controlled. In a particular embodiment, an MMP9-specific antibody or antigen-binding fragment thereof according to the invention is administered systemically or locally.

In a particular embodiment, a MMP9-specific antibody or antigen-binding fragment thereof according to the invention is administered by the subcutaneous or intravenous route.

The dosage administered, as a single or multiple dose, to an individual will vary depending on a variety of factors, including the pharmacokinetic properties, condition and characteristics of the patient (sex, age, body weight, health, height), degree of symptoms, treatments concurrent, frequency of treatment and desired effect.

Commonly, therapeutically effective amounts of an active pharmaceutical antibody range from 1 mg to 150 mg / kg of body weight. If the regimen is a continuous infusion, it may be in the range of 0.250 mg to 20 mg per kg of body weight.

In particular, effective therapeutic amounts of an active pharmaceutical antibody range from 1 mg to 50 mg / kg of body weight. If the regimen is a continuous infusion, it may be in the range of 0.250 mg to 13 mg per kg of body weight.

Patients

Commonly, patients according to the invention are patients suffering from an MMP9-related disease, in particular an MMP9-related disease selected from the group consisting of inflammatory and autoimmune diseases, cancers or tumors, lung diseases, fibrotic lung diseases, septicemia , muscular dystrophy, allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and diseases eye disease, or any other disease or disorder related to MMP9. In one embodiment, patients according to the invention are patients suffering from an inflammatory and / or autoimmune disease including, for example, inflammatory bowel diseases (IBD) including Crohn's disease (CD), ulcerative colitis (UC ), indeterminate colitis, collagenous colitis, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, polymyositis, atherosclerosis.

In a particular embodiment, patients according to the invention are patients suffering from inflammatory bowel disease, more particularly penetrating and constrictive complications of Crohn's disease and ulcerative colitis.

In another embodiment, the patients according to the invention are patients suffering from a lung disease including asthma, fibrotic lung diseases such as idiopathic pulmonary disease, chronic obstructive pulmonary disease (COPD), or a disease selected from septicemia, muscular dystrophy. , allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and eye diseases.

In another embodiment, patients according to the invention are patients suffering from a cancer or tumor including, for example, hematopoietic cancer, brain cancer, breast cancer, colorectal cancer, head and neck cancer, pancreatic cancer, lung cancer, ovarian cancer, urinary bladder cancer, liver cancer, melanoma, prostate cancer, muscle cancer, mesenchymal cancer, esophagogastric adenocarcinoma, non-small cell lung cancer, squamous cell carcinoma of the lung, adenocarcinoma of the lung, gastric adenocarcinoma, pancreatic adenocarcinoma and hepatocellular carcinoma.

In a particular embodiment, the patients according to the invention are patients suffering from colorectal cancer or adenocarcinoma.

In another embodiment, patients according to the invention are patients suffering from a disease selected from the group consisting of inflammatory and autoimmune diseases, inflammatory bowel diseases, cancers or tumors, fibrotic diseases, cardiovascular diseases, neuropsychiatric disorders or eye diseases. .

In another embodiment, the patients according to the invention are patients suffering from fibrotic diseases, eg, systemic sclerosis, multifocal fibrosclerosis, graft-versus-host scleroderma disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, pulmonary fibrosis, fibrosis. liver, kidney fibrosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus.

In another embodiment, the patients according to the invention are patients suffering from eye diseases, for example fibrotic pathologies of the lens, corneal diseases, diabetic retinopathy, "dry" or "wet" age-related macular degeneration. , proliferative vitreoretinopathy, cataract formation, pterygium, keratoconus, age-related macular degeneration and diabetic retinopathy.

In another embodiment, patients according to the invention are patients suffering from cardiovascular diseases, for example hypertension, atherosclerosis, myocardial infarction, heart failure or coronary artery disease, hypertension, pulmonary hypertension, pulmonary valve disease or tricuspid, aortic and mitral valve disease, aortic coarctation, atherosclerosis, myocardial infarction, heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, chronic arrhythmia, cardiac fibrosis, and coronary artery disease.

In another embodiment, patients according to the invention are patients suffering from neurological disorders, eg, amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, neuronal inflammation, cerebral ischemia and neuropathic pain.

Uses and methods according to the invention

MMP9-specific antibody or antigen-binding fragment thereof, nucleic acids, vectors, host cells, compositions according to the invention are for use in the diagnosis, prevention or treatment of disorders associated with, caused by, or accompanied by elevated MMP9 levels and / or elevated MMP9 activity.

In one embodiment, an MMP9-specific antibody or antigen-binding fragment thereof is provided according to the invention for use as a medicament.

Another embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of an MMP9-related disease selected from the group consisting of inflammatory and autoimmune diseases, cancers or tumors, lung diseases, diseases fibrotic lung disease, septicemia, muscular dystrophy, allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and eye diseases, or any other disease or disorder related to MMP9.

Another embodiment provides an antibody or fragment thereof according to the invention, for use in the prevention and / or treatment of an MMP9-related disease selected from the group consisting of inflammatory and autoimmune diseases, inflammatory bowel diseases, cancers or tumors, fibrotic diseases, cardiovascular diseases, neuropsychiatric disorders or eye diseases. Another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of a disease selected from inflammatory and autoimmune diseases, inflammatory bowel diseases, cancers or tumors, fibrotic diseases, cardiovascular diseases , neurological disorders or eye diseases.

Another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of an inflammatory and / or autoimmune disease, in particular inflammatory bowel diseases (IBD) including Crohn's disease (CD) , ulcerative colitis (UC), indeterminate colitis, collagenous colitis, rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, polymyositis, atherosclerosis.

Another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of a lung disease that includes asthma, fibrotic lung diseases such as idiopathic pulmonary disease, chronic obstructive pulmonary disease (COPD), or a disease selected from septicemia, muscular dystrophy, allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and eye diseases.

Still another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of a cancer or tumor, in particular a cancer selected from the group consisting of hematopoietic cancer, brain cancer, cancer of breast, head and neck cancer, pancreatic cancer, ovarian cancer, urinary bladder cancer, lung cancer, liver cancer, melanoma, prostate cancer, muscle cancer, mesenchymal cancer, esophagogastric adenocarcinoma, non-small cell lung cancer , lung squamous cell carcinoma, lung adenocarcinoma, gastric adenocarcinoma, pancreatic adenocarcinoma, hepatocellular carcinoma, more particularly colorectal cancer or adenocarcinoma. Still another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of a fibrotic disease, in particular systemic sclerosis, multifocal fibrosclerosis, graft versus host scleroderma disease in bone marrow transplant recipients. , nephrogenic systemic fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus.

Yet another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of an ocular disease, in particular fibrotic pathologies of the lens, corneal diseases, diabetic retinopathy, macular degeneration related to age, “dry” or “wet”, proliferative vitreoretinopathy, cataract formation, pterygium, keratoconus, age-related macular degeneration and diabetic retinopathy.

Still another particular embodiment provides an antibody or fragment thereof, according to the invention, for use in the prevention and / or treatment of a cardiovascular disease, in particular hypertension, pulmonary hypertension, pulmonary or tricuspid valve disease, disease of the mitral and aortic valve, aortic coarctation, atherosclerosis, myocardial infarction, heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, chronic arrhythmia, cardiac fibrosis, and coronary artery disease.

Still another particular embodiment provides an antibody or fragment thereof according to the invention for use in the prevention and / or treatment of a neurological disorder, in particular amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, neuronal inflammation, cerebral ischemia and pain. neuropathic.

In another embodiment it is provided the use of a specific antibody for MMP9 or binding fragment antigen thereof according to the invention for the preparation of a pharmaceutical composition for preventing and / or treating a disease related with ^m MP9 selected from the group consisting of in inflammatory and autoimmune diseases, cancers or tumors, lung diseases, fibrotic lung diseases, sepsis, muscular dystrophy, allergy, kidney fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes, and eye diseases, or any other MMP9-related disease or disorder.

In a specific embodiment there is provided the use of a specific antibody for MMP9 or antigen-binding fragment thereof according to the invention for the preparation of a pharmaceutical composition for the prevention and / or treatment of inflammatory and autoimmune diseases, inflammatory diseases of the intestine, cancers or tumors, fibrotic diseases, cardiovascular diseases, neuropsychiatric disorders or eye diseases.

In a particular embodiment, the use of an antibody specific for MMP9 or antigen-binding fragment thereof according to the invention is provided for the preparation of a pharmaceutical composition for the treatment and / or treatment of an inflammatory and / or autoimmune disease in an individual, particularly for the prevention or treatment of inflammatory bowel diseases (IBD) including Crohn's disease (CD), ulcerative colitis (UC), indeterminate colitis, collagenous colitis, rheumatoid arthritis (RA), multiple sclerosis (MS) , systemic lupus erythematosus (SLE), Sjogren's syndrome, systemic sclerosis, polymyositis, atherosclerosis.

In a specific embodiment the use of a specific antibody for MMP9 or antigen-binding fragment thereof according to the invention is provided for the preparation of a pharmaceutical composition for the prevention and / or treatment of inflammatory bowel disease, in particular complications Penetrating and constrictive diseases of Crohn's disease and ulcerative colitis.

In a particular embodiment the use of an antibody specific for MMP9 or antigen-binding fragment thereof according to the invention is provided for the preparation of a pharmaceutical composition for the prevention and / or treatment of a lung disease including asthma, diseases fibrotic lung diseases such as idiopathic pulmonary disease, chronic obstructive pulmonary disease (COPD), or a selected disease of septicemia, muscular dystrophy, allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and eye diseases .

In an alternative embodiment the use of a MMP9-specific antibody or antigen-binding fragment thereof according to the invention is provided for the preparation of a pharmaceutical composition for the prevention and / or treatment of a cancer or tumor, in particular for the prevention and / or treatment of a cancer selected from the group consisting of hematopoietic cancer, brain cancer, breast cancer, head and neck cancer, pancreatic cancer, ovarian cancer, urinary bladder cancer, lung cancer, cancer of liver, melanoma, prostate cancer, muscle cancer, mesenchymal cancer, esophagogastric adenocarcinoma, non-small cell lung cancer, squamous cell lung carcinoma, lung adenocarcinoma, gastric adenocarcinoma, pancreatic adenocarcinoma, hepatocellular carcinoma, more particularly colorectal cancer or adenocarcinoma.

In a specific embodiment there is provided the use of an MMP9-specific antibody or antigen-binding fragment thereof according to the invention, for the preparation of a pharmaceutical composition for the prevention and / or treatment of colorectal cancer or adenocarcinoma.

In a specific embodiment the use of a specific antibody for MMP9 or antigen-binding fragment thereof according to the invention is provided for the preparation of a pharmaceutical composition for the prevention and / or treatment of a fibrotic disease, in particular sclerosis systemic, multifocal fibrosclerosis, graft-versus-host scleroderma disease in bone marrow transplant recipients, nephrogenic systemic fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus.

In a specific embodiment, the use of a specific antibody for MMP9 or antigen-binding fragment thereof according to the invention is provided for the preparation of a pharmaceutical composition for the prevention and / or treatment of an ocular disease, in particular pathologies fibrotic lens, corneal disease, diabetic retinopathy, age-related macular degeneration, "dry" or "wet", proliferative vitreoretinopathy, cataract formation, pterygium, keratoconus, age-related macular degeneration and diabetic retinopathy.

In a specific embodiment a specific antibody for MMP9 or antigen-binding fragment thereof according to the invention are useful for the preparation of a pharmaceutical composition for the prevention and / or treatment of a cardiovascular disease, in particular hypertension, pulmonary hypertension , pulmonary or tricuspid valve disease, aortic and mitral valve disease, coarctation of the aorta, atherosclerosis, myocardial infarction, heart failure, ischemic cardiomyopathy, dilated cardiomyopathy, chronic arrhythmia, cardiac fibrosis, and coronary artery disease.

In a specific embodiment a specific antibody for MMP9 or antigen-binding fragment thereof according to the invention are useful for the preparation of a pharmaceutical composition for the prevention and / or treatment of a neurological disorder, in particular amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis, neuronal inflammation, cerebral ischemia, and neuropathic pain.

In another embodiment the antibodies of the invention are useful in a method for the prevention and / or treatment of an MMP9-related disease selected from the group consisting of inflammatory and autoimmune diseases, cancers or tumors, lung diseases, fibrotic lung diseases, septicemia, muscular dystrophy, allergy, renal fibrosis, scleroderma, dilated cardiomyopathy, Chagas disease, cardiovascular diseases, neuropsychiatric disorders, diabetes and eye diseases, or any other disease or disorder related to MMP9.

When used herein "biological sample" refers to cells, tissue samples or cellular components (such as cell membranes or cellular components) obtained from an individual, in particular from an individual suspected of having, or having suffer from, inflammatory or autoimmune disease and / or cancer or tumor or are at high risk of developing such a disorder.

Examples of a biological sample include samples of blood, serum, plasma, cerebrospinal fluid, synovial fluid, urine, feces, and tissue including cells isolated from such tissue. Tissue samples include formalin-fixed or frozen tissue sections.

Any suitable method for the detection and analysis of MMP9 may be employed, including diagnostic assay techniques known in the art such as competitive binding assay, direct or indirect sandwich assays, and immunoprecipitation assays performed on heterogeneous or homogeneous phases.

In a particular embodiment, the invention provides an ex vivo method to detect the presence and / or concentration of the MMP9 protein in a biological sample, comprising the steps of:

(i) provide a biological sample from an individual,

(ii) reacting said biological sample with at least one antibody or antigen-binding fragment thereof according to the invention, under conditions sufficient to bind the MMP9 protein present in said biological sample to said at least one antibody or fragment thereof through antigen-antibody interactions; Y

(iii) detect a signal proportional to the level of the antigen-antibody complex formed in step (ii), where the intensity of the signal correlates with the concentration of the MMP9 protein in the biological sample.

In a particular embodiment, the invention provides an ex vivo method for detecting the presence and / or concentration of active MMP9 protein in a biological sample, wherein at least one antibody or antigen-binding fragment thereof according to the invention comprises a variable region of the heavy chain of SEQ ID NO: 19 and a variable region of the light chain of ID S ^and C NO: 70.

More specifically, an ex-vivo method of prognosis or diagnosis useful for an inflammatory and / or autoimmune disease or cancer associated with an elevated level of MMP9 from a biological sample from an individual is provided, comprising the steps of:

(a) provide a biological sample from an individual;

(b) putting said biological sample in contact with a solid matrix where at least one antibody or fragment thereof according to the invention binds to, where the contact is made under conditions sufficient to bind the MMP9 protein present in said sample biological fluid to said at least one antibody or fragment thereof through antigen-antibody interactions;

(c) removing any unbound MMP9 protein from the surface of said solid matrix;

(d) detect a signal proportional to the level of antigen-antibody complex bound to said solid matrix, (e) compare the signal level detected in step (d) with the signal level detected under the same conditions with a negative control,

wherein a level of signal detected in the individual's sample that is greater than the level of signal detected in the negative control is indicative of an elevated level of MMP9 associated with an inflammatory and / or autoimmune disease or cancer.

Kit

The antibodies of the invention can be used as a kit comprising at least one antibody or antigen-binding fragment thereof according to the invention, and / or at least one nucleic acid encoding said antibody or fragment thereof, and / or at least one vector comprising said nucleic acid, and / or at least one host cell according to the invention, and optionally instructional material.

In a particular embodiment, the kit comprises at least one antibody or antigen-binding fragment thereof according to the invention, which is to be coupled or is already coupled to a solid matrix.

Examples of suitable solid matrix for the invention include any suitable solid phase support to carry out an immunoassay or method, such as beads, microparticles, nanoparticles, tubes, tissues. or plates, films, slides, wells, formed of or coated with glass, polystyrene, polypropylene, nitrocellulose, quartz, ceramic, dextran or other materials. For example, the solid matrix is in the form of microtiter wells, such as a 96-well microtiter plate.

Attachment of the antibodies, or fragments thereof, to the solid matrix in a kit can be accomplished by adsorption or chemical coupling to a solid phase support. Any means known in the art can be used to immobilize a protein or peptide to a solid support. Antibodies or fragments of these according to the invention can be covalently or non-covalently attached to the solid matrix by techniques such as covalent attachment through an amide or ester bond or adsorption. Peptides can be linked using binding pairs such as biotin and avidin or antibody and antigen. After fixing the peptides to the solid matrix, the solid matrix can be incubated with a blocking solution (containing a blocking protein such as bovine serum albumin) to reduce the non-specific adsorption of antibodies in a test sample to the surface. of support. According to one aspect, the antibodies or fragment thereof according to the invention can be synthesized directly on the solid matrix of the kit.

According to one aspect, when the kit comprises at least one antibody or fragment thereof, according to the invention, or a combination of these for coupling to a solid matrix as a solid phase support, the kit further, as an option, it comprises coupling reagents and / or a solid matrix to perform an immunoassay.

According to another aspect, the kit further comprises at least one rinse reagent for washing unbound material prior to detection in order to avoid detection of background noise. Commonly rinse reagents comprise standard buffers known in the art.

The present invention is defined by the appended claims

The invention having been described, the following examples are presented by way of illustration, and not by limitation.

EXAMPLES

Example 1: Generation and isolation of the anti-MMP9 antibodies according to the invention

The anti-MMP9 antibodies according to the invention were obtained by carrying out the following steps: 1 / Hit Discovery (HD) phage display

A ScFv library was used as a source of ScFv fragments. This library is of natural origin (that is, it was constructed using the PBMC of healthy, non-immunized human donors), of modest size (approximately 2,510 ⁹ VH / VL combinations) and contains VH domains derived from the IgM repertoire without clonal selection and without class switching. Using this library, ScFv candidates were first selected based on their binding to human or mouse MMP9 (full-length, pro-, and activated forms) by phage display, ScFv fragments were reformatted into IgG1 and selected for neutralization. functional activity of MMP9 (in particular, by determining the IC ⁵⁰ , species selectivity, neutralization modality (proMMP9 or MMP9). This step allows the identification of 10 candidate antibodies, which could be divided into two mechanistic classes : those that block MMP9 activity by interfering with the activation of latent proMMP9 (suggesting that the pro-domain is part of the epitope recognized by antibodies) and those that directly interfere with the catalytic activity of activated MMP9 (suggesting that the CAT_fn domain is part of the epitope recognized by antibodies).

2 / Hit Optimization (HO) by exchanging the VL chain for affinity maturation:

Four of the HD candidates obtained in the previous step were subjected to Hit Optimization through the exchange of the lambda light chain to improve their potency. Basically, the VH chains of the parental HD clone were permuted against the entire lambda VL content of the original library, generating several specific libraries of new clones.

3 / Optimization of the leader:

Two HO candidates (F20-VH / B03-VLc and F20-VH / B08-VLc) were selected based on their mechanism of action, that is, the inhibition of the enzymatic activation of MMP9, for optimization and characterization of the best candidate. (leader), through, in particular (i) optimization by framework residue germination (as defined by Kabat) to minimize deviation from the closest human germline sequence to potentially improve physical properties and reduce the risk of immunogenicity, and (ii) the change of some amino acids or amino acid motifs within the CDRs that could lead to chemical instability or aggregation of antibodies. This strategy allows the generation of antibodies that comprise the VH / VL regions indicated in Table 2 above.

In the following examples, the specific VH / VL regions mentioned in Table 3 below were reformatted as human IgG4 for further characterization. For this purpose, nucleic acids encoding the VH and VL chains of interest were cloned into pTT vectors (engineered for cloning compatibility with the original strands) for transient co-expression of IgG4 in HEK293 cells.

Table 3

Example 2: Potency and efficacy of some anti-MMP9 antibodies according to the invention in activity assays of human MMP3 / MMP9 and human MMP9

The functional neutralization of the catalytic activity of MMP9 towards the fluorogenic polymer substrate DQ-gelatin was evaluated for some antibodies according to the invention.

The signature activity of MMP9 (and MMP2) is gelatin degradation. Gelatin is an essential and irreversibly denatured form derived from various collagens, several of which are considered key physiological substrates for MMP9. Unlike small peptide substrates, the binding and recognition of gelatin (similar to collagen) by MMP9 is complex and known to be mediated through regions of the molecule other than the active site, including the fibronectin and PEX domains. In order to preserve these physiologically important 'exosite' interactions and facilitate the isolation of potential 'nonclassic' allosteric neutralizing classes, a fluorogenically quenched soluble gelatin polymer (DQ-gelatin) was chosen as the key substrate for screening and sorting purposes. based on plates. The hydrolysis of the substrate was mediated by the truncated fragment of MMP9 (ie, pro-domain deleted). The test was conducted in two modes. The first mode, termed the "MMP9 assay," used pre-activated MMP9 (from engineered MMP9 containing a LETD caspase 8 recognition motif at the native cleavage junction) in which the pro-domain was first removed via the targeted caspase 8 cleavage. The second mode, termed the "MMP3 / MMP9 assay", was devised to incorporate pro-domain removal (ie, the activation step) as an additional binding process within the assay. This was achieved by combining the catalytic domain of human MMP3 (Calbiochem, 444217 5) and full-length proMMP9 (latent form) in the test cocktail together with the substrate DQ-gelatin (Lubio Science, D 12054) and antibodies to be tested. MMP3, considered to be a physiologically relevant activator of MMP9, has been shown to remove the pro-domain through a two-step sequential mechanism ( Ogata et al, 1992, J. Biol. Chem. 267 ( 6): 3581-4) , and is frequently co-expressed with MMP9 in diseased tissues. Importantly, activated MMP3 itself does not appear to significantly cleave the DQ-gelatin substrate. Therefore, DQ-gelatin hydrolysis (fluorescence emission) in this assay is dependent on MMP3-mediated activation of MMP9 and the intrinsic catalytic activity of MMP9, which offers the potential to characterize neutralizers that interfere with any of the processes.

Therefore, the "MMP3 / MMP9 assay" was used to determine the ability of anti-MMP9 antibodies to neutralize proMMP9 activation and its downstream catalytic activity. Briefly, the recombinant human proMMP9 aliquots were pre-incubated with various dilutions of the antibodies to be tested for one hour. Digestion was started by adding a recombinant catalytic domain from human MMP3, together with the MMP9-specific fluorescent substrate, DQ-Gelatin. The fluorescence signal emitted (Excitation at 485 nm, Emission at 520 nm) is proportional to the digestion of the gelatin substrate, as a consequence of the catalytic activity of the mature MMP9 enzyme. The fluorescence signal was plotted against antibody concentrations and maximum mean inhibitory potencies (IC ⁵⁰ values) were deduced from the non-linear regression curves.

The "MMP9 assay" was used to determine the ability of anti-MMP9 antibodies to directly neutralize the catalytic activity of mature MMP9. In summary, the assay is similar to the "MMP3 / MMP9 assay" but uses a catalytically active, caspase-cleaved recombinant MMP9 in place of proMMP9, therefore the addition of the recombinant catalytic domain of human MMP3 is not required.

All antibodies according to the invention that were tested were able to effectively decrease the digestion of DQ-gelatin in both enzyme assays. This result indicates that all antibodies according to the invention block the activation of proMMP9 for the catalytically active enzyme and / or block the catalytic activity of MMP9 (Table 4, Figure 6).

Table 4 . Potency and efficacy of anti-MMP9 antibodies to neutralize human proMMP9 processing and / or its downstream catalytic activity on gelatin

The F20-VH-GL1-V4-V9 / B08-VL-GL6c antibody according to the invention was compared to Comparative Antibody 1 (antibody known as AB0041, which has a heavy chain amino acid sequence of SEQ ID NO: 44 and light chain SEQ ID NO: 45) using the highly purified, activated MMP9 "MMP9 assay". Only the F20-VH-GL1-V4-V9 / B08-VL-GL6c antibody according to the invention effectively decreased DQ-gelatin digestion with an IC ⁵⁰ of 2.6 nM, while Comparative Antibody 1 was inactive. This result indicates that the F20-VH-GL1-V4-V9 / B08-VL-GL6c antibody according to the invention is an inhibitor of the enzymatic activity of MMP9, while the comparative antibody 1 is not.

In the "MMP3 / MMP9 assay", the F20-VH-GL1-V4-V9 / B08-VL-GL6c antibody according to the invention and the comparative antibody 1 effectively decreased DQ-gelatin digestion with an IC ⁵⁰ of 9.52 nM and 0.20 nM, respectively. Overall, the results of these two enzyme assays indicate that Comparative Antibody 1 is an inhibitor of MMP9 activation, while F20-VH-GL1-V4-V9 / B08-VL-GL6c antibody is an inhibitor of MMP9 activity. catalytic MMP9 and possibly an inhibitor of MMP9 activation. Example 3: Species specificity of some anti-MMP9 antibodies according to the invention in the MMP3 / MMP9 assay

The species specificity of the enzyme inhibitory activities of the anti-MMP9 antibodies was evaluated using the "MMP3 / MMP9 assay" described in Example 2. The enzyme used was Cynomologus monkey (cyno), rat or mouse proMMP9.

Some antibodies according to the invention, which were tested, were able to effectively block the activation and / or the catalytic activity upstream of Cynomologus monkey (cyno), rat and mouse proMMP9 (Tables 5, 6 and 7, respectively).

Table 5 . Potency and efficacy of anti-MMP9 antibodies to neutralize Cynomolgus monkey proMMP9 in MMP3 / MMP9 gelatin assays.

Table 6 . Potency and efficacy of anti-MMP9 antibodies to neutralize rat proMMP9 in MMP3 / MMP9 gelatin assays.

Table 7 . Potency and efficacy of anti-MMP9 antibodies to neutralize mouse proMMP9 or active MMP9 in gelatin assays of MMP3 / MMP9 and m Mp9, respectively.

NO: Not detected

Example 4: MMP selectivity of some anti-MMP9 antibodies according to the invention in the assay of catalytic activity of human MMPs

The selectivity of anti-MMP9 antibodies was evaluated in an assay evaluating MMP-mediated cleavage of a fluorogenic peptide substrate, OmniMMP ™ RED (Enzo, BML-P277-9090), using the MMP Inhibitor Profiling Kit (Enzo, BML- AK308). In summary, aliquots of the recombinant catalytic domain of various human MMPs (MMP1, MMP2, MMP3, MMP8, MMP9, MMP12, MMP13, MMP14 and MMP19) were pre-incubated with a fixed concentration (100 nM) of antibodies to be tested for one hour. . The MMP inhibitor NNGH (Enzo, BML-PI115-9090) and anti-MMP2 / 9 (539A-M0237-D02, comparative antibody 3, which has a heavy chain amino acid sequence of SEQ ID NO: 64 and light chain SEQ ID NO: 65), anti-MMP9 (539A-M0240-B03, comparative antibody 2, having a heavy chain amino acid sequence of SEQ ID NO: 46 and light chain SEQ ID NO: 47) and isotype control antibodies (IgG1 anti-HEL) were included in the test, as positive and negative controls. Then fluorogenic peptide substrate was added and fluorescence was measured. The fluorescence signal emitted is proportional to the digestion of the peptide substrate, therefore, to the activity of the catalytic domains of MMP. The fluorescence signal in the absence of the inhibitor was normalized to 100% activity. The signal obtained in the presence of each inhibitor was given relative to this value and referred to the percentage of the remaining activity.

All antibodies according to the invention, which were tested, were able to effectively block the catalytic activity of human MMP9, while they had no significant effect on the catalytic activity of the other human MMPs tested (Figure 7). All antibodies according to the invention also had no significant effect on the catalytic activity of human MMP7, MMP10, MMP16 and MMP17 (data not shown).

Example 5: Potency of the anti-MMP9 antibody according to the invention in activity assays using natural secreted forms of human MMP9 neutrophils

Several natural forms of human MMP9 have been described (monomeric MMP9, dimeric MMP9 and MMP9 bound to the lipocalin form NGAL associated with neutrophil gelatinase B (also called lipocalin-2), the latter form also called NGAL / MMP9 ( Rudd et al. ., 1999, Biochemistry, 38, 13937-13950)) and was shown to be associated with disease states.

Additional characterization of anti-MMP9 antibodies of the invention includes neutralization of natural forms of MMP9 derived from neutrophils. For this purpose, the functional neutralization of the neutrophil-derived MMP9 catalytic activity towards the fluorogenic substrate OmniMMP ™ RED (from ENZO) was evaluated by antibodies according to the invention.

Activation of the proMMP9 fraction in neutrophil-derived MMP9 preparations was performed by pre-treatment with APMA (p-aminophenyl mercuric acetate), which makes the catalytic site of MMP9 accessible, prior to the functional neutralization assay with the antibody variant anti-MMP9 F20-VH / B08-VLc of the invention (F20-VH-GL1-V4-V9 / B08-VL-GL6c). In summary, aliquots of various APMA-activated neutrophil-derived human forms of proMMP9 (monomeric, dimeric, and NGAL / MMP9) were incubated with antibody to rehearse for an hour. The isotype control antibody (IgG4 anti-HEL) was included in the test, as a negative control. Then fluorogenic peptide substrate was added and fluorescence was measured. The emitted fluorescence signal (Excitation at 540 nm, Emission at 590 nm) is proportional to the digestion of peptide substrate, therefore to the activity of the catalytic domains of MMP.

As shown in Figure 8, the F20-VH / B08-VLc antibody variant of the invention (F20-VH-GLl-V4-V9 / B08-VL-GL6c) neutralizes the activity of multiple physiological forms of human MMP9: Monomeric MMP9, dimeric MMP9 and NGAL / MMP9 with 100% efficiency.

The antibody of the invention was compared with comparative antibody 1 using the "MMP3 / MMP9 assay" described in Example 2. The variant of the F20-v H / B08-VLc antibody of the invention (F20-VH-GLl-V4- V9 / B08-VL-GL6c) inhibits the enzymatic activity of MMP9 derived from human neutrophils with 100% efficacy, either in monomeric or dimeric form or bound to NGAL (Figure 9), while comparative antibody 1 only showed partial inhibition (25% efficacy) over all forms of neutrophil-derived m MP9 (Figure 9). Complete neutralization of all naturally-occurring active forms of MMP9 by the F20-VH / B08-VLc antibody variant of the invention (F20-VH-GL1-V4-V9 / B08-VL-GL6c) should result in superior efficacy in patients. with high serum levels of monomeric, dimeric and / or complex NGAL / MMP9 MMP9.

Example 6: Kinetics of binding and affinity for the pro and activated forms of human MMP9 of the anti-MMP9 antibodies according to the invention

The binding of anti-MMP9 antibodies to recombinant human MMP9 antigen was characterized using the normal Surface Plasmon Resonance (SPR) assay. In a first step, the anti-MMP9 antibodies were captured by the anti-human Fc antibody immobilized on a BIAcore sensor chip. In a second step, MMP9 activated with pro-MMP9 or MMP3 was titrated from 4.7 to 150 nM in 2-fold dilutions. Sensorgrams revealed that both F20-VH / B08-VLc variants (F20-VH-GL1-V4-V9 / B08-VL-GL6c) and Comparative Antibody 1 bound well to MMP3-activated MMP9 (Figure 10). Comparative antibody 1 also bound well to pro-MMP9, but minimal signal was observed with the F20-VH / b08-VLc antibody variant (F20-VH-GL1-V4-V9 / B08-VL-GL6c) (Figure 10 ). Table 8 presents the binding affinities (KD) of the F20-VH / B08-VLc antibody variant according to the invention (F20-VH-GL1-V4-V9 / B08-VL-GL6c) and of the comparative antibody 1 towards MMP9 activated with pro-human MMP9 and MMP3. Since less than 1% binding was observed at an antibody concentration of 150 nM, the KD of the F20-VH / B08-VLc antibody variant (F20-VH-GL1-V4-V9 / B08-VL-GL6c) for pro-MMP9 it was estimated to be greater than 15 pM.

Table 8

KD - equilibrium dissociation constant, all calculated from the curves Example 7: Mapping of the epitopes of anti-MMP9 antibodies according to the invention.

Another characterization of the anti-MMP9 antibodies of the invention includes the identification of the regions of human MMP9 that are important for the binding of the antibody, and thus the epitopes are defined. This characterization was carried out on one member of each of the F20 / B08 and F20 / B03 antibodies, namely, F20-VH-GL1-V1-V9-V14 / B03-VL-GL1 c and F20-VH-GL1- V4-V9 / B08-VL-GL6c.

The technology used to determine MMP9 epitopes capable of specifically binding to the anti-MMP9 antibodies of the invention applies chemical crosslinking and mass spectrometry ( Peter and Tomer, 2001, Anal. Chem., 73, 4012-4019; Pimenova et al, 2008, J. Mass Spectrom. JMS, 43, 185-195; Herzog et al., 2012, Science 337, 1348-1352). First, the antibodies of the invention and pro-MMP9 were mixed in solution and subjected to chemical cross-linking using specially designed deuterated cross-linkers. The covalently linked MMP9 and the antibody-MMP9 complex were then subjected to proteolysis using 3 different proteolytic enzymes in order to generate a large number of overlapping peptides that covered the entire sequence of the MMP9. Peptides from MMP9 alone and from the cross-linked antibody-MMP9 complex were analyzed by high resolution mass spectrometry (nLC-Orbitrap MS) and compared to determine the interacting peptides of the immune complex.

This analysis showed that the interaction interface between human MMP9 (SEQ ID NO 1) and the antibodies of the invention is located within 3 discontinuous MMP9 epitopes that include the following MMP9 amino acid sequences:

SEQ ID NO: 41: 150AVTPLTFTRVYSRDADIVIQF170 (corresponding to amino acid positions 150 to 170 of human MMP9) (referred to herein as Region 1),

SEQ ID NO: 42: 198IQGDAHFDDDELWSLGKGWVPTRFG223 (corresponding to amino acid positions 198 to 223 of human MMP9) (referred to herein as region 2), and

SEQ ID NO: 43: 419MYPMYRFTEGPPLHKDDVNGIR440 (corresponding to amino acid positions 419 to 440 of human MMP9) (referred to herein as region 3).

Various recombinant mutants of human MMP9 were used to determine amino acids important for the binding of comparative antibody 1 to MMP9 (mouse monoclonal AB0041, WO 2013/130078). Analysis identified residues E111, D113, R162 and 1198 as important for the binding of comparative antibody 1 to human MMP9. Two of these amino acids, R162 and 1198, are present in regions of the epitope recognized by the anti-MMP9 antibodies of the invention (region 1 and region 2). However, region 3 defines a novel region of the epitope specifically important for the anti-MMP9 antibodies of the invention. Region 3 is within the catalytic domain of MMP9. Since the anti-MMP9 antibodies of the invention, unlike comparative antibody 1, are capable of inhibiting the enzymatic activity of fully mature MMP9, whereas comparative antibody 1 does not bind fully human MMP9, it is possible that Binding to region 3 confers the anti-MMP9 antibodies of the invention the ability to neutralize the enzymatic activity of fully mature MMP9.

Furthermore, the mouse monoclonal antibody REGA-3G12 ( Martens et al., Supra) was shown to bind to the G171 to L187 peptide of human MMP9 using binding and competition assays. The region of MMP9 (G171-L187) bound by the REGA-3G12 antibody is outside of the 3 regions of MMP9 recognized by the anti-MMP9 antibodies of the invention.

Example 8: Direct binding and competitive binding of anti-MMP9 antibodies to pro and activated forms of human MMP9

Capabilities direct binding of anti-MMP9 MMP9 to pro-human and human activated MMP9 MP3 ^m, respectively, were evaluated using a standard ELISA protocol. Briefly, 96-well plates were coated with (2.5 pg / mL) of any of the MMP9 forms for 16 h at room temperature. After 2 h of incubation with blocking buffer (2% goat serum in PBS / Tween) and four washings with wash buffer (PBS / Tween 0.05% v / v), various concentrations of biotinylated anti-MMP9 antibodies (comparative antibody 1 or variant F20-VH / B08-VLc (F20-VH-GLl-V4-V9 / B08-VL-GL6c) were added in duplicate and incubated for 1 h. The wells were then washed four times and the antibodies were revealed. biotinylates bound using streptavidin coupled with HRP and TMB substrate. Absorbance was read at 450 nm and 620 nm for each duplicate and data were expressed as corrected absorbance (A450-A620). Results shown in Figure 11 indicate that, both comparative antibody 1 and anti-MMP9 antibodies, the F20-VH / B08-VLc variant (F20-VH-GL1-V4-V9 / B08-VL-GL6c), bind strongly to MMP3-activated MMP9 (Figure 11B ), in a dose-dependent manner.In contrast, while comparative antibody 1 binds well to pro-MMP9 , in a dose-dependent form (Figure 11A), the anti-MMP9 antibody, the F20-VH / B08-VLc variant (F20-VH-GLl-V4-V9 / B08-VL-GL6c), only shows significant binding to pro-MMP9 at the highest concentration tested (5 pg / mL) (Figure 11A).

To analyze if the antibody, the F20-VH / B08-VLc variant (F20-VH-GLl-V4-V9 / B08-VL-GL6c), could compete with comparative antibody 1 for binding to MMP9, the same was used ELISA assay with MMP3-activated MMP9 coating, but the F20-VH / B08-VLc variant of purified antibodies or comparative antibody 1 were added for 2 hours before the addition of the biotinylated anti-MMP9 antibodies. After four washes with wash buffer, a predefined optimal dose of biotinylated comparative antibody 1 or F20-VH / B08-VLc was added and incubated for 1 h. The results shown in Figure 11 panel C indicate that the addition of the variant anti-MMP9 antibody F20-VH / B08-VLc (F20-VH-GL1-V4-V9 / B08-VL-GL6c) does not prevent the binding of the antibody comparative 1 to MMP9, while the same concentration of purified comparative antibody 1 completely prevents further binding of biotinylated comparative antibody 1 to MMP9.

Taken together, these binding experiments show that, unlike comparative antibody 1, the variant F20-VH / B08-VLc (F20-VH-GLl-V4-V9 / B08-VL-GL6c) anti-MMP9 antibody binds preferentially to the active form of MMP9 over pro-MMP9, and which does not compete with Comparative Antibody 1 for binding to MMP9.

Example ^{9 :} Effect of the anti-MMP9 antibody according to the invention in cancer cell invasion assay

Cancer cell line invasion was assessed using Matrigel-coated Transwell assays. The inserted transwell membranes (provided in the Cultrex kit - 3455-024) were coated with basement membrane extract (BME x0.5; provided in the Cultrex kit) and incubated for 24h at 37 ° C. Meanwhile, MGC803 (human gastric cancer cell line - Easy-Bio, China) were harvested in RPMI culture medium (Gibco - 52400025) and were serum starved (nutrient-free medium). The cells were then recovered and resuspended at 0.5 x 106 cells / mL in nutrient-free medium containing 100 ng / mL of PMA (phorbol 12-myristate 13-Sigma-P8139 acetate) and either 25 pM MMP inhibitor (GM -6001 from Millipore - CC1000), 10 pg / mL anti-MMP9 antibody (F20-VH / B08-VLc variant F20-VH-GL1-V4-V9 / B08-VL-GL6c), 10 pg / mL anti -IgG4 (isotype control) or none (medium). One hundred pL of treated cells were seeded in a Matrigel-coated transwell and 500 pL of RPMl medium containing 10% FCS was added on the lower side of the transwell. The plates were incubated for 16 h at 37 ° C. Invasive cells were quantified by incubation of the lower part of the transwell membrane with Calcein-AM, whose cleavage into fluorescent calcevna is proportional to the cell counts. The emitted fluorescent signal (Excitation at 485 nm, emission at 520 nm) reflects the efficiency of cell invasion through Matrigel.

The results show that the anti-MMP-9 F20-VH / B08-VLc variant antibody (F20-VH-GL1-V4-V9 / B08-VL-GL6c) efficiently inhibits the migration of cancer cells MGC803 through Matrigel with similar efficacy when compared to the MMP inhibitor GM-6001 (Figure 12). As metastatic cells escape from their place of origin invading surrounding tissue, including basement membranes, this result indicates that the anti-MMP9 of the invention could be an effective anti-migratory and anti-invasive drug for metastatic cancers.

Example 10: Activity of the anti-MMP9 antibody according to the invention in a mouse colitis model

The mouse dextran sulfate sodium (DSS) -informed colitis model of inflammatory bowel disease (IBD) is widely used in preclinical studies. Drugs that are approved for the treatment of ulcerative colitis, such as steroids, metronidazole, 5'-aminosalicylates, cyclosporine, and anti-TNFa immunotherapy have shown efficacy in reducing the severity of the disease in the DSS model showing that this model is a Relevant model for the translation of mouse human disease data ( Perse et al., 2012, J. Biomed. Biotechnol, 2012: 718617). Mouse colitis is induced by adding DSS to drinking water resulting in damage to the lining of the colon. The clinical manifestation of acute phase DSS colitis may include weight loss and diarrhea. The typical histological changes of acute DSS colitis are similar to those seen in human IBD and include mucin depletion, epithelial degeneration, and necrosis leading to the disappearance of epithelial cells. The latter is accompanied by infiltration of neutrophils within the lamina propria and submucosa, cryptitis, crypt abscesses, and inflammation of the mucosa and submucosa of the colon.

The therapeutic efficacy of an anti-MMP9 antibody according to the invention was evaluated in a model of DSS-induced colitis in BALB / c mice. Colitis was induced with 4% (w / v) DSS in drinking water for 5 days. The 30 mg / kg doses of the F20-VH / B08-VLc (variant F20-VH-GL1-V4-V9 / B08-VL-GL6c) or isotype control (IgG4) antibodies were injected intraperitoneally in groups of 10 mice on day 6, 9 and 12, and the animals were sacrificed on day 14.

Disease course was assessed in masked mode by video endoscopy of the lower colon on days 6, 10, and 14. Colitis was visually scored on a 0-14 scale based on degree of ulceration, vascularization , and granularity present in the tissue and length of the colon involved as follows: ulceration (0, 1, 2 or 3), masking of vascularization (0, 1, 2 or 3), granularity (0, 1, 2 or 3), erythema (0, 1, 2 or 3), implied length (0, 1: localized, 2: diffuse). Each mouse was assigned a unique endoscopy score corresponding to the damage observed along the entire length of the examined colon. Animals that had an endoscopic score <5 on day 6, prior to starting antibody treatment, were judged to have insufficient disease severity and were excluded from the other analysis. As shown in Figure 13, on day 6, both groups had similar mean endoscopy scores while at the end of the study on Day 14, the animals treated with F20-VH / B08-VLc-showed a significant improvement (p = 0.005 ) in the mean endoscopy score compared to the isotype control antibody treated group.

At the completion of the study (day 14), the distal colon of each mouse was excised, fixed with formalin and embedded in paraffin and sectioned for histology. All sections were stained with hematoxylin and eosin and examined in a blinded mode for treatment groups. Tissues were scored for epithelial damage and inflammatory cell infiltration according to the 1-4 scoring scale for each parameter. The histology score represents the sum of the scores for epithelial damage and inflammatory cell infiltration, and therefore ranges from 0 to 8. The results are shown in Figure 14.

Mice treated with DSS developed colitis characterized by epithelial hyperplasia and goblet cell depletion, infiltration of inflammatory cells in the mucosa and submucosa. Crypt abscesses and mucosal erosion and ulceration were also observed in some cross sections of the colon. As shown in Figure 13, analysis of histological scores showed that anti-MMP9 antibody treatment was associated with a decrease in mean F20-VH / B08-VLc colitis severity score compared to control group treated with isotype antibody. Similarly, the mean scores for inflammatory cell infiltration and epithelial damage also decreased by anti-MMP9 antibody treatment when compared to the control group treated with isotype antibody.

Example 11: Activity of an anti-MMP9 antibody according to the invention in a mouse model of heterotopic transplantation of intestinal fibrosis

Severe damage to mucosal tissue is a main feature of inflammatory bowel disease (IBD). Tissue injury is a trigger for repair activities by surrounding cells. Rapid wound closure is important to reduce the time during which the barrier function of the intestinal wall deteriorates, but excessive tissue repair promotes fibrosis, a common occurrence in Crohn's disease (CD). Fibrosis gives rise to the formation of stenosis in 10-40% of patients ( Cosnes et al., 2002, Inflamm. Bowel Dis., 8 ( 4): 244-50; Freeman, 2003, J. Clin. Gastroenerol, 37 ( 3): 216-9), an indication for surgery in approximately 80% of patients with stenosis ( Cosnes et al, 2002, supra).

The heterotopic transplantation of intestinal resections in mice gives rise to the disappearance of the intestinal epithelium and culminates in the fibrotic occlusion of the intestinal lumen, recapitulating the histological and molecular characteristics of human intestinal fibrosis, such as thickening of the luminal wall, exaggerated deposition of collagen, and expression of profibrotic mediators.

Donor small intestine resections (C57BL / 6-Tg UBC-GFP mouse labeled with green fluorescent protein) were transplanted subcutaneously into the neck of recipient C57BL / 6 mice. Mice were injected intraperitoneally on day 5, 8 and 10 with 30 mg / kg of an anti-MMP9 antibody of the invention (F20-VH / B08-VLc, variant F20-VH-GL1-V4-V9 / B08 -VL-GL6c), or isotype control antibody (IgG4), 5 mice per group. Small intestine grafts were explanted 14 days after transplantation. After each explant, each graft was divided into three equal segments. The central segment was fixed in 4% formalin, and prepared for histopathological evaluation. The two outer segments were flash frozen in liquid nitrogen and stored at -70 ° C until RNA or protein extraction.

Histological cross sections were stained with Sirius red to highlight collagen (red spot). The thickness of the collagen layer was determined by an independent researcher masked for the type of experiment. Microscopic evaluation was performed using an AxioCam MRc5 on a Zeiss Axiophot microscope. The thickness of the collagen layer was measured with Axio Vision Release 4.7.2 software (Zeiss). Thickness was calculated from eight locations in representative areas (in eight investigated samples for each time point) at 100-fold magnification. Statistical analyzes were performed using PRISM 6 software. The unpaired t-test was used for comparison between groups.

Compared to the control isotype, anti-MMP9 treatment with F20-VH / B08-VLc significantly protects against loss of epithelial structure in heterotopic intestinal grafts as shown in Figure 15 and significantly reduces (p <0.0001) the thickness of the collagen layer in heterotopic intestinal grafts, as shown in Figure 16. This result indicates that the anti-MMP9 antibody of the invention could effectively (lacuna) fibrotic diseases and fistulizing Crohn's disease.

Example 12: Effect of anti-MMP9 antibody in nude Swiss mice bearing HCT-116 subcutaneous human colon tumor cells.

The human colon cancer cell line HCT-116 (1x10 7 cells) was injected subcutaneously into the right flank of nude mice and grown to "100 mm3 before starting treatment. The anti-MMP9 antibody of the invention F20-VH / B08-VLc, variant F20-VH-GLl-V4-V9 / B08-VL-GL6c or control IgG4 isotype antibody are injected intraperitoneally for 3 weeks. Mice received a loading dose of antibody at 50 mg / kg on the first day of treatment and then dosed twice weekly at 30 mg / kg. Mice were sacrificed on day 21 after the start of treatment and tumors and primary organs with metastases were harvested for analysis. The weight and size of the primary tumor were measured and the number of visually metastatic foci in the lymph nodes, liver, lung and peritoneum were counted.

Claims (1)

An isolated antibody specific for MMP9, or an antigen-binding fragment thereof, wherein said antibody or fragment thereof comprises: (i) a heavy chain variable region comprising an amino acid sequence of SEQ ID NO: 11, or a variant of SEQ ID NO: 11, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 11: N54Q, N54R, N54K, N54H, D62E, S63T, M116I, D88A and V93L and (ii) further comprising a light chain variable region selected from the following group: (1) SEQ ID NO: 24, or a variant of SEQ ID NO: 24, wherein the amino acid at position 107 of SEQ ID NO: 24 is substituted with the substitution: V107L; (2) SEQ ID NO: 67, or a variant of SEQ ID NO: 67, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions: V45L, V70A and V107L; (3) SEQ ID NO: 29, or a variant of SEQ ID NO: 29, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 29: RsA , V18I, L49M, T62N, L81Q, and A83E; Y (4) SEQ ID NO: 69, or a variant of SEQ ID NO: 69, wherein at least one amino acid is substituted by a different amino acid selected from the following substitutions in the sequence of SEQ ID NO: 69: RsA , V18I, L49M, T62N, L81Q, A83E and FssY. The isolated antibody or fragment thereof according to any of claims 2 to 3, wherein said variable region of the heavy chain comprises an amino acid sequence selected from: (i) the amino acid sequence SEQ ID NO: 12 (ii) the amino acid sequence SEQ ID NO: 13 (iii) the amino acid sequence SEQ ID NO: 14 (iv) the amino acid sequence SEQ ID NO: 15 (v) the amino acid sequence SEQ ID NO: 16 (vi) the amino acid sequence SEQ ID NO: 17 (vii) the amino acid sequence SEQ ID NO: 18 (viii) the amino acid sequence SEQ ID NO: 19 (ix) the amino acid sequence SEQ ID NO: 20, (x) the amino acid sequence of SEQ ID NO: 11. The isolated antibody or fragment thereof according to any of claims 1 and 2, wherein the variable region of the light chain comprises an amino acid sequence selected from: (i) the amino acid sequence of SEQ ID NO: 67; (ii) the amino acid sequence of SEQ ID NO: 68; (iii) the amino acid sequence of SEQ ID NO: 69; Y (iv) the amino acid sequence of SEQ ID NO: 70; The isolated antibody or fragment thereof according to any of claims 1 to 3, wherein a variable region of the heavy chain comprises an amino acid sequence selected from: (i) the amino acid sequence SEQ ID NO: 12 (ii) the amino acid sequence SEQ ID NO: 13 (iii) the amino acid sequence SEQ ID NO: 14 (iv) the amino acid sequence SEQ ID NO: 15 (v) the amino acid sequence SEQ ID NO: 16 (vi) the amino acid sequence SEQ ID NO: 17 (vii) the amino acid sequence SEQ ID NO: 18 (viii) the amino acid sequence SEQ ID NO: 19 (ix) the amino acid sequence SEQ ID NO: 20: and (x) the amino acid sequence of SEQ ID NO: 11 wherein a variable region of the light chain comprises an amino acid sequence selected from: (xi) the amino acid sequence SEQ ID NO: 24: (xii) the amino acid sequence SEQ ID NO: 25: (xiii) the amino acid sequence SEQ ID NO: 29: (xiv) the amino acid sequence SEQ ID NO: 30: (xv) the amino acid sequence SEQ ID NO: 57: (xvi) the amino acid sequence SEQ ID NO: 58: (xvii) the amino acid sequence SEQ ID NO: 59: (xviii) the amino acid sequence SEQ ID NO: 60: (xix) the amino acid sequence SEQ ID NO: 61: (xx) the amino acid sequence SEQ ID NO: 62: (xxi) the amino acid sequence SEQ ID NO: 63: (xxii) the amino acid sequence SEQ ID NO: 67: (xxiii) the amino acid sequence SEQ ID NO: 68: (xxiv) the amino acid sequence SEQ ID NO: 69: and (xxv) the amino acid sequence SEQ ID NO: 70: The isolated antibody or fragment thereof, according to any of the preceding claims, wherein a variable region of the heavy chain comprises an amino acid sequence with (a), and a variable region of the light chain comprises an amino acid sequence with (b) selected from combinations of the group consisting of: (b): the amino acid sequence SEQ ID NO: 24, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 24, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 25, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 25, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 29, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 29, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 30, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 30, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 57, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 57, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 58, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 58, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 59, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 59, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 60, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 60, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 61, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 61, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 62, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 62, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 63, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 63, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 67, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 14, 15, 16, 17. 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 67, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 55 and 56; (b): the amino acid sequence SEQ ID NO: 68, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 68, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56; (b): the amino acid sequence SEQ ID NO: 69, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; (b): the amino acid sequence SEQ ID NO: 69, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56; (b): the amino acid sequence SEQ ID NO: 70, and (a): the amino acid sequence selected from SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20; Y (b): the amino acid sequence SEQ ID NO: 70, and (a): the amino acid sequence selected from SEQ ID NO: 48, 53, 54, 55 and 56. 6. The isolated antibody according to any one of claims 1 to 9, wherein said antibody comprises a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 70. 7. The isolated antibody or fragment thereof according to any of claims 1 to 6, further comprising a human constant region, in particular of an IgG isotype, such as an IgG4 isotype comprising a human heavy chain constant region of SEQ ID NO: 40, and a constant region of the human light chain of SEQ ID NO: 66. 8. The isolated antibody according to any of claims 1 to 8, which is a human monoclonal antibody. An antibody or fragment thereof according to any one of claims 1 to 8 for use as a medicament. 10. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8. 11. A recombinant expression vector comprising a nucleic acid molecule according to claim 10. 12. A host cell comprising a recombinant vector according to claim 11. 13. A pharmaceutical composition comprising one or more of the following: (i) an MMP9-specific antibody or antigen-binding fragment thereof according to any of claims 1 to 8, (ii) a nucleic acid according to claim 10, (iii) a vector according to claim 11, and / or (iv) a host cell according to claim 12, and at least one pharmaceutically acceptable carrier. The antibody or fragment thereof according to any of claims 1 to 8, for use in the prevention and / or treatment of a disease selected from inflammatory and autoimmune diseases, inflammatory bowel diseases, cancers or tumors, fibrotic diseases, cardiovascular diseases, neurological disorders or eye diseases. 15. The antibody or fragment thereof for use according to claim 14, wherein the inflammatory bowel disease is Crohn's disease, in particular fistulizing Crohn's disease. 16. An ex-vivo method to detect the presence and / or concentration of the MMP9 protein in a biological sample, comprising the steps of: (i) provide a biological sample from an individual, (ii) reacting said biological sample with at least one antibody or antigen-binding fragment thereof according to any of claims 1 to 8, under conditions sufficient to bind the MMP9 protein present in said biological sample to said at least one antibody or fragment thereof through antigen-antibody interactions; Y (iii) detect a signal proportional to the level of the antigen-antibody complex formed in step (ii), where the intensity of the signal correlates with the concentration of the MMP9 protein in the biological sample.